Nucleotide sequences of lats genes

ABSTRACT

The present invention relates to a tumor suppressor gene, termed large tumor suppressor (lats), and methods for identifying tumor suppressor genes. The method provides nucleotide sequences of lats genes, and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. In a specific embodiment, the lats protein is a human protein. The invention further relates to fragments (and derivatives and analogs thereof) of lats which comprise one or more domains of a lats protein. Antibodies to lats, its derivatives and analogs, are additionally provided. Methods of production of the lats proteins, derivatives and analogs, e.g., by recombinant means, are also provided. Therapeutic and diagnostic methods and pharmaceutical compositions are provided. The invention also relates to recombinant plants and animals and methods of increasing the growth of edible plants and animals. In specific examples, isolated lats genes, from Drosophila, mouse, and human, and the sequences thereof, are provided.

The present application is a divisional application of application Ser.No. 08/411,111 now U.S. Pat. 5,994,503, filed Mar. 27, 1995, which isincorporated by reference herein its entirety.

1. INTRODUCTION

The present invention relates to tumor suppressor genes, in particularto “lats” genes (large tumor suppressor) and their encoded proteinproducts, as well as derivatives and analogs thereof. Production of latsproteins, derivatives, and antibodies is also provided. The inventionfurther relates to therapeutic compositions and methods of diagnosis andtherapy.

2. BACKGROUND OF THE INVENTION

Tumorigenesis in humans is a complex process involving activation ofoncogenes and inactivation of tumor suppressor genes (Bishop, 1991, Cell64:235-248). Tumor suppressor genes in humans have been identifiedthrough studies of genetic changes occurring in cancer cells (Ponder,1990, Trends Genet. 6:213-218; Weinberg, 1991, Science 254:1138-1146).In Drosophila, tumor suppressor genes have been previously identified byrecessive overproliferation mutations that cause late larval and pupallethality (Gateff, 1978, Science 200:1448-1459; Gateff and Mechler,1989, CRC Crit. Rev. Oncogen 1:221-245; Bryant, 1993, Trends Cell Biol.3:31-35; Török et al., 1993, Genetics 135:71-80). Mutations of interestwere identified when dissection of dead larvae and pupae revealedcertain overproliferated tissues. Several genes identified in homozygousmutants have been cloned including 1(1)discs large-1(dlg; Woods andBryant, 1991, Cell 66:451-464; Woods and Bryant, 1993, Mechanisms ofDevelopment 44:85-89), fat (Mahoney et al., 1991, Cell 67:853-868),1(2)giant larvae (lgl. Lützelschwab et al., 1987, EMBO J. 6:1791-1797;Jacob et al., 1987, Cell 50:215-225), expanded (ex; Boedigheimer andLaughon, 1993, Development 118:1291-1301; Boedigheimer et al., 1993,Mechanisms of Development 44:83-84), hyperplastic discs (hyd; Mansfieldet al., 1994, Developmental Biology 165:507-526) and the gene encodingthe S6 ribosomal protein (Watson et al., 1992, Proc. Natl. Acad. Sci.USA 89:11302-11306; Stewart and Denell, 1993, Mol. Cell. Biol.13:2524-2535).

Although examining homozygous mutant animals has allowed the successfulidentification of overproliferation mutations that cause late larval andpupal lethality, mutations that cause lethality at early developmentalstages are unlikely to be recovered by this approach. The presentinvention solves this problem by providing a method for identifyingtumor suppressor genes that does not exclude genes that when mutatedcause lethality in early developmental stages, and provides genes thusidentified with a fundamental role in regulation of cell proliferation.

Citation of references hereinabove shall not be construed as anadmission that such references are prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences of lats genes(Drosophila, human, and mouse lats and lats homologs of other species),and amino acid sequences of their encoded proteins, as well asderivatives (e.g., fragments) and analogs thereof. Nucleic acidshybridizable to or complementary to the foregoing nucleotide sequencesare also provided. In a specific embodiment, the lats protein is a humanprotein.

The invention also relates to a method of identifying tumor suppressorgenes that does not exclude from identification genes that causelethality at early developmental stages, thus overcoming the limitationsof prior art methods. The method thus allows the identification of genesthat regulate cell proliferation and that act at early developmentalstages. The genes which thus can be identified play a fundamental rolein regulation of cell proliferation such that their dysfunction (e.g.,by lack of expression or mutation) leads to overproliferation andcancer.

Lats is a gene provided by the present invention, identified by themethod of the invention, that acts to inhibit cell proliferation, andthat plays a crucial role throughout development.

The invention also relates to lats derivatives and analogs of theinvention which are functionally active, i.e., they are capable ofdisplaying one or more known functional activities associated with afull-length (wild-type) lats protein. Such functional activities includebut are not limited to kinase activity, antigenicity [ability to bind(or compete with lats for binding) to an anti-lats antibody],immunogenicity (ability to generate antibody which binds to lats), andability to bind (or compete with lats for binding) to a receptor/ligandfor lats (e.g., a SH3 domain-containing protein).

The invention further relates to fragments (and derivatives and analogsthereof) of lats which comprise one or more domains of a lats protein.

Antibodies to lats, and lats derivatives and analogs, are additionallyprovided.

Methods of production of the lats proteins, derivatives and analogs,e.g., by recombinant means, are also provided.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on lats proteins and nucleic acids. Therapeuticcompounds of the invention include but are not limited to lats proteinsand analogs and derivatives (including fragments) thereof; antibodiesthereto; nucleic acids encoding the lats proteins, analogs, orderivatives; and lats antisense nucleic acids.

The invention provides for treatment of disorders of overproliferation(e.g., cancer and hyperproliferative disorders) by administeringcompounds that promote lats activity (e.g., lats, an agonist of lats;nucleic acids that encode lats).

The invention also provides methods of treatment of disorders involvingdeficient cell proliferation (growth) or in which cell proliferation isotherwise desired (e.g., degenerative disorders, growth deficiencies,lesions, physical trauma) by administering compounds that antagonize,(inhibit) lats function (e.g., antibodies, antisense nucleic acids).

Antagonizing lats function can also be done to grow larger animals andplants, e.g., those used as food or material sources.

Animal models, diagnostic methods and screening methods forpredisposition to disorders, and methods to identify lats agonists andantagonists, are also provided by the invention.

3.1. DEFINITIONS

As used herein, underscoring or italicizing the name of a gene shallindicate the gene, in contrast to its encoded protein product which isindicated by the name of the gene in the absence of any underscoring oritalicizing. For example, “lats” shall mean the lats gene, whereas“lats” shall indicate the protein product of the lats gene.

4. DESCRIPTION OF THE FIGURES

FIG. 1. (A-B) Identifying overproliferation mutations in mosaic flies.(A) Although animals that are homozygous for a lethal mutation could dieat an early developmental stage, mosaic flies carrying clones of cellsthat are homozygous for the same mutation could live. One can identifypotential tumor suppressors by generating and examining clones ofoverproliferated mutant cells in mosaic animals. The geneticconstitution of these mosaic flies is similar to the mosaicism of thetumor patients. (B) Genetic scheme. The P-element insertions carryingthe FLP recombinase (hsFLP; Golic and Lindquist, 1989, Cell 59:499-509),its target site, FRT (solid arrows, Xu and Rubin, 1993, Development117:1223-1237), the yellow⁺ and mini-white⁺ marker genes (y⁺ andmini-w⁺, open arrows) are indicated. Mutagenized males were crossed tofemales to produce heterozygous embryos. Clones of cells homozygous forthe induced mutations were generated in developing first-instar larvaeby mitotic recombination at the FRT sites induced with the FLPrecombinase. Mosaic adults were examined for overproliferated mutantpatches (w⁻, y⁻). Individuals carrying clones of interest were thenmated to recover the mutations of interest in the next generation (Xuand Rubin, 1993, Development 117:1223-1237; Xu and Harrison, 1994;Methods in Cell Biology 44:655-682). Clones of ommatidia derived fromfast proliferating mutant cells were identified since they were largerthan their darkly pigmented wt (wild-type) twin-spot clones(mini-w⁺/mini-w⁺).

FIG. 2. (A-L) Mutant phenotypes. (A) A clone of unpatterned,overproliferated lats mutant cells in the eye. (B) Induced at the samestage, the 93B mutant cells formed a less overproliferated clone. (C) Athird instar lats^(e26-1) larva (right) was much larger than a wtsibling (left; at 18° C.). (D) Wing discs from the larva in (C) (wt,top; lats^(e26-1), bottom). (E) Dissected central nervous systems (wt,top; lats^(e26-1), bottom). (F) A SEM (scanning electron microscope)view of a lats clone near the eye. (G) A closer view of a region in (F)showing the irregularity of the sizes and shapes of the mutant cells.(H) A plastic section of a mutant clone similar to the one in (F). Cellsseem to be “budding” out of the surface to form new proliferating lobes(arrows). (I) A lats clone on the back. The boxed area is shown in (J).The bristles in the mutant clone are short, bent and often split(arrows). (K) A closer view of the hairs in a lats clone on the bodyshowing enlarged bases and bent tips. (L) A section of a lats clone onthe back showing extra cuticle deposits (arrows). All the mutant cloneswere induced with lats^(x1) unless stated differently.

FIG. 3. Organization of the Drosophila lats gene. The genomicrestriction map of the lats region is aligned with the lats 5.7 kbtranscript unit. The direction of transcription is indicated with largearrows. The sizes of the lats introns are as follows: intron 1 (5.0 kb),intron 2 (5.8 kb), intron 3 (68 bp), intron 4 (63 bp), intron 5 (64 bp),intron 6 (61 bp), intron 7 (62 bp). The genomic DNA from +7.5 (BglII) to−4.2 (EcoRI) was used to screen a total imaginal disc cDNA library,which isolated three groups of cDNAs: lats, T1, T2. The introns in theT2 transcript are not labeled. Only parts of the zfh-1 (Fortini et al.,1991, Mechan. Dev. 34:113-122) and T1 transcripts are indicated. Thelocations of the P-element insertion (lats^(P1)), the deletions in thefive excision alleles (lats^(e7-2, e78, e100, e119, e148)) and inlats^(a1), lats^(a4) are indicated at the bottom. The slash indicates agap in the genomic map. Restriction sites: EcoRI (small open arrow),BglII (open box) and BamHI (open circle). The BglII site at the −0.5position of the CLT-52 clone is not present in other genomic DNA. Ascale is labeled under the restriction map.

FIG. 4. RNA blot analysis of the Drosophila lats mRNA. Five μg ofpoly(A)⁺RNA isolated from various developmental stages was separated ona 1% agarose gel, and hybridized with ³²P-labeled 5′ end 1 kb probe fromthe Drosophila lats cDNA. E0-2 hrs, E2-4 hrs, E4-6 hrs, E6-8 hrs, E8-16hrs and E16-24 hrs indicate the age of the embryos in hours. RNA fromfirst, second and third instar larvae is denoted by L1, L2, and L3,respectively. The numbers and arrows on the right correspond to the sizeand location of the RNA standards. A 5.7 kb RNA was found in all thedevelopmental stages, whereas a 4.7 kb RNA was predominantly present in0 to 4 hour old embryos. The blot was also hybridized with DNA from theribosomal protein gene, RNAl.

FIG. 5. Composite cDNA sequence of the Drosophila lats gene. The entirecDNA sequence (SEQ ID NO:1) corresponding to the 5.7 kb lats RNA isshown. This nucleotide sequence is a composite of two cDNA clones(nucleotide 1-191 from cDNA 9 and the rest from cDNA A2). The sequenceof the corresponding genomic DNA has been determined and is identical tothe cDNA sequence except where indicated (above the cDNA sequence). Thepredicted amino acid sequence (SEQ ID NO:2) is shown below the CDNAsequence. The opa repeat is indicated by the heavy bar. The location ofthe putative SH3 binding site and the RERDQ peptides are designated bydashed lines. The two sites that match the polyadenylation signalconsensus sequence are underlined. The second site is located at 12 bpaway from the 3′ end of the cDNA. The locations of the introns areindicated by vertical arrows. The underlined 141 bp sequence at the 3′end of the lats transcript is identical to the 5′ end untranslatedsequence of the class I transcript of the Drosophila phospholipase Cgene, plc-21. The location of the 446 bp deletion in the lats^(a1)allele is also indicated.

FIG. 6. (A-C) Schematic of the Drosophila lats predicted protein (SEQ IDNO:2) and the related proteins (A) and sequence comparison of theproteins homologous to lats (B). In FIG. 6A, solid, hatched, open andshaded boxes denote putative SH3 binding site, opa repeat, RERDQ peptideand kinase domain in the lats protein, respectively. The Dbf20, Dbf2 andCOT-1 proteins are illustrated at the bottom. The regions that arehomologous to lats are indicated by shaded boxes. The degrees ofsequence similarity (percentage of identical sequences insideparentheses; percentage of identical or conservative substitutionsoutside parentheses) between lats and the three related proteins areindicated above the corresponding regions of these proteins. In FIG.6B-C, the carboxy-terminal half of lats is compared to the six mostrelated proteins that are revealed by blastp (a software program thatsearches for protein sequence homologies) search as of Sep. 1, 1994.Neurospora cot-1 (SEQ ID NO:11); tobacco PKTL7 (SEQ ID NO:12); commonice plant protein kinase (SEQ ID NO:13); spinach protein kinase (SEQ IDNO:14); yeast Dbf-20 (SEQ ID NO:15); yeast Dbf2 (SEQ ID NO:16). Aminoacid residues identical to lats are highlighted. Numbers at thebeginning of every sequence refer to the position of that amino acidwithin the total protein sequence. The boundary of the kinase domain isdefined according to Hanks et al. (1988, Science 241:42-52). Thelocation of a region of about 40 amino acid residues that is notconserved among the proteins is indicated by the heavy bar above thesequence. The sequence of PKTL7 from tobacco, Nicotiana tabacum, wassubmitted to Genbank by Huang,Y. (X71057). Both the sequence of theprotein kinase from spinach, Spinacia oleracea, and the sequence of theprotein kinase from common ice plant, Mesembryanthemum crystallinum,were submitted to Genbank by Baur, B., Winter, K., Fischer, K. andDietz, K., (Z30329 and Z30330).

FIG. 7. cDNA sequence (SEQ ID NO:5) and deduced protein sequence (SEQ IDNO:6) of a mouse lats homolog, m-lats.

FIG. 8. cDNA sequence (SEQ ID NO:7) and deduced protein sequence (SEQ IDNO:8) of a mouse lats homolog, m-lats2.

FIG. 9. cDNA sequence (SEQ ID NO:3) and deduced protein sequence (SEQ IDNO:4) of a human lats homolog, h-lats.

FIG. 10. Schematic diagram of plasmid pBS(KS)-h-lats, containing thefull length coding sequence of the h-lats cDNA.

FIG. 11. Alignment of the h-lats protein sequence (SEQ ID NO:4) (uppercase letters) with the m-lats protein sequence (SEQ ID NO:6) (lower caseletters). A dot indicates amino acid identity; a dash indicates adeletion relative to the sequence on the line above. The amino-terminalportion of the m-lats protein is not shown due to the missing 5′ end ofthe m-lats cDNA coding region.

FIG. 12. Alignment of the h-lats protein sequence (SEQ ID NO:4) (uppercase letters) with the m-lats2 protein sequence (SEQ ID NO:8) (lowercase letters). A dot indicates amino acid identity; a dash indicates adeletion relative to the sequence on the line above. The amino-terminalportion of the m-lats2 protein is not shown due to the missing 5′ end ofthe m-lats2 cDNA coding region.

FIG. 13. Alignment of the h-lats protein sequence (SEQ ID NO:4) (uppercase letters) with the Drosophila lats protein sequence (SEQ ID NO:2)(lower case letters). A dot indicates amino acid identity; a dashindicates a deletion relative to the sequence on the line above.Insertions in the Drosophila sequence relative to the human sequence areindicated below the sequence line. Conserved domains are indicated.LSD2=lats-split domain 2; LSD2a=LSD2 anterior portion; LSD2p=LSD2posterior portion. The putative SH3-binding domain and the kinase domainare shown. LSD1 =lats split domain 1; LSD1a=LSD1 anterior portion;LSD1p=LSD1 posterior portion. LFD=lats flanking domain. LCD1=latsC-terminal domain 1; LCD2=lats C-terminal domain 2; LCD3=lats C-terminaldomain 3.

FIG. 14. Schematic diagram of plasmid pCaSpeR-hs-h-lats, an expressionvector containing the full length coding sequence of the h-lats cDNA.

FIG. 15. Northern blot analysis of h-lats expression in normal humantissues. A ³²P-labeled BamHI fragment of h-lats was used as a probe forhybridization to polyA⁺ RNA from the normal human fetal and adulttissues indicated for each lane. The positions of standard molecularweight markers are shown at right. The positions of the h-lats RNA andof β-actin RNA (used as a standard) are shown.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of lats genes, andamino acid sequences of their encoded proteins. The invention furtherrelates to fragments and other derivatives, and analogs, of latsproteins. Nucleic acids encoding such fragments or derivatives are alsowithin the scope of the invention. The invention provides lats genes andtheir encoded proteins of many different species. The lats genes of theinvention include Drosophila, human, and mouse lats and related genes(homologs) in other species. In specific embodiments, the lats genes andproteins are from vertebrates, or more particularly, mammals. In apreferred embodiment of the invention, the lats genes and proteins areof human origin. Production of the foregoing proteins and derivatives,e.g., by recombinant methods, is provided.

The invention also relates to a method of identifying tumor suppressorgenes that does not exclude from identification genes that causelethality at early developmental stages, thus overcoming the limitationsof prior art methods. The method thus allows the identification of genesthat regulate cell proliferation and that act at early developmentalstages. The genes which thus can be identified play a fundamental rolein regulation of cell proliferation such that their dysfunction (e.g.,due to lack of expression or mutation) leads to overproliferation andcancer.

Lats is a gene provided by the present invention, identified by themethod of the invention, that acts to inhibit cell proliferation, andthat plays a crucial role throughout development.

The invention also relates to lats derivatives and analogs of theinvention which are functionally active, i.e., they are capable ofdisplaying one or more known functional activities associated with afull-length (wild-type) lats protein. Such functional activities includebut are not limited to kinase activity, antigenicity [ability to bind(or compete with lats for binding) to an anti-lats antibody],immunogenicity (ability to generate antibody which binds to lats),ability to bind (or compete with lats for binding) to anSH3-domain-containing protein or other ligand, ability to inhibit cellproliferation, tumor inhibition, etc.

The invention further relates to fragments (and derivatives and analogsthereof) of lats which comprise one or more domains of the lats protein.

Antibodies to lats, its derivatives and analogs, are additionallyprovided.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on lats proteins and nucleic acids and anti-latsantibodies. The invention provides for treatment of disorders ofoverproliferation (e.g., cancer and hyperproliferative disorders) byadministering compounds that promote lats activity (e.g., lats proteinsand functionally active analogs and derivatives (including fragments)thereof; nucleic acids encoding the lats proteins, analogs, orderivatives, agonists of lats).

The invention also provides methods of treatment of disorders involvingdeficient cell proliferation or in which cell proliferation (growth) isotherwise desirable (e.g., growth deficiencies, degenerative disorders,lesions, physical trauma) by administering compounds that antagonize, orinhibit, lats function (e.g., antibodies, lats antisense nucleic acids,lats derivatives that are dominant-negative protein kinases).

Inhibition of lats function can also be done to grow larger farm animalsand plants.

Animal models, diagnostic methods and screening methods forpredisposition to disorders are also provided by the invention.

The invention is illustrated by way of examples infra which disclose,inter alia, the cloning and characterization of D. melanogaster lats(Section 6); the cloning and characterization of mouse and human latshomologs (Section 7); the sequence and domain conservation among thelats homologs (Section 8); the functional interchangeability of thehuman and Drosophila lats homologs (Section 9); and the differentiallydecreased expression of human lats in human tumor cell lines (Section10).

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

5.1. ISOLATION OF THE LATS GENES

The invention relates to the nucleotide sequences of lats nucleic acids.In specific embodiments, lats nucleic acids comprise the cDNA sequencesof SEQ ID NO:1, 3, 5, or 7, or the coding regions thereof, or nucleotidesequences acids encoding a lats protein (e.g., a protein having thesequence of SEQ ID NO:2, 4, 6, or 8). The invention provides purifiednucleic acids consisting of at least 8 nucleotides (i.e., a hybridizableportion) of a lats sequence; in other embodiments, the nucleic acidsconsist of at least 25 (continuous) nucleotides, 50 nucleotides, 100nucleotides, 150 nucleotides, or 200 nucleotides of a lats sequence, ora full-length lats coding sequence. In another embodiment, the nucleicacids are smaller than 35, 200 or 500 nucleotides in length. Nucleicacids can be single or double stranded. The invention also relates tonucleic acids hybridizable to or complementary to the foregoingsequences. In specific aspects, nucleic acids are provided whichcomprise a sequence complementary to at least 10, 25, 50, 100, or 200nucleotides or the entire coding region of a lats gene. In a specificembodiment, a nucleic acid which is hybridizable to a lats nucleic acid(e.g., having sequence SEQ ID NO:3 or 7), or to a nucleic acid encodinga lats derivative, under conditions of low stringency is provided. Byway of example and not limitation, procedures using such conditions oflow stringency are as follows (see also Shilo and Weinberg, 1981, Proc.Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA arepretreated for 6 h at 40° C. in absolution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations arecarried out in the same solution with the following modifications: 0.02%PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol)dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 40° C., and thenwashed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and reexposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

In another specific embodiment, a nucleic acid which is hybridizable toa lats nucleic acid under conditions of high stringency is provided. Byway of example and not limitation, procedures using such conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C. in prehybridization mixture containing 100μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe.Washing of filters is done at 37° C. for 1 h in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C. for 45 min before autoradiography. Otherconditions of high stringency which may be used are well known in theart.

In another specific embodiment, a nucleic acid, which is hybridizable toa lats nucleic acid under conditions of moderate stringency is provided(see, e.g., Section 7.2).

Nucleic acids encoding derivatives and analogs of lats proteins (seeSections 5.6 and 5.6.1), and lats antisense nucleic acids (see Section5.8.2.2.1) are additionally provided. As is readily apparent, as usedherein, a “nucleic acid encoding a fragment or portion of a latsprotein” shall be construed as referring to a nucleic acid encoding onlythe recited fragment or portion of the lats protein and not the othercontiguous portions of the lats protein as a continuous sequence.

Fragments of lats nucleic acids comprising regions conserved between(with homology to) other lats nucleic acids, of the same or differentspecies, are also provided. Nucleic acids encoding one or more latsdomains are provided.

Specific embodiments for the cloning of a lats gene, presented as aparticular example but not by way of limitation, follows:

For expression cloning (a technique commonly known in the art), anexpression library is constructed by methods known in the art. Forexample, mRNA (e.g., human) is isolated, CDNA is made and ligated intoan expression vector (e.g., a bacteriophage derivative) such that it iscapable of being expressed by the host cell into which it is thenintroduced. Various screening assays can then be used to select for theexpressed lats product. In one embodiment, anti-lats antibodies can beused for selection.

In another embodiment, polymerase chain reaction (PCR) is used toamplify the desired sequence in a genomic or cDNA library, prior toselection. Oligonucleotide primers representing known lats sequences canbe used as primers in PCR. In a preferred aspect, the oligonucleotideprimers represent at least part of the lats conserved segments of stronghomology between lats of different species (e.g., LCD1, LCD2, kinasedomain, LFD, SH3 binding domain, LSD1, and LSD2 domains; see, e.g.,Section 8 infra.) The synthetic oligonucleotides may be utilized asprimers to amplify by PCR sequences from a source (RNA or DNA),preferably a cDNA library, of potential interest. PCR can be carriedout, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp™). The DNA being amplified can include mRNA or cDNAor genomic DNA from any eukaryotic species. One can choose to synthesizeseveral different degenerate primers, for use in the PCR reactions. Itis also possible to vary the stringency of hybridization conditions usedin priming the PCR reactions, to allow for greater or lesser degrees ofnucleotide sequence similarity between the known lats nucleotidesequence and the nucleic acid homolog being isolated. For cross specieshybridization, low stringency conditions are preferred. For same specieshybridization, moderately stringent conditions are preferred. Aftersuccessful amplification of a segment of a lats homolog, that segmentmay be molecularly cloned and sequenced, and utilized as a probe toisolate a complete cDNA or genomic clone. This, in turn, will permit thedetermination of the gene's complete nucleotide sequence, the analysisof its expression, and the production of its protein product forfunctional analysis, as described infra. In this fashion, additionalgenes encoding lats proteins and lats analogs may be identified.

The above-methods are not meant to limit the following generaldescription of methods by which clones of lats may be obtained.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the lats gene. The nucleic acid sequencesencoding lats can be isolated from vertebrate, mammalian, human,porcine, bovine, feline, avian, equine, canine, as well as additionalprimate sources, insects, plants, etc. The DNA may be obtained bystandard procedures known in the art from cloned DNA (e.g., a DNA“library”), by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired cell. (See,for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U. K. Vol. I, II.) Clones derived from genomic DNAmay contain regulatory and intron DNA regions in addition to codingregions; clones derived from cDNA will contain only exon sequences.Whatever the source, the gene should be molecularly cloned into asuitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, if an amount of a portion of a lats (of any species)gene or its specific RNA, or a fragment thereof (see Section 5.6), isavailable and can be purified and labeled, the generated DNA fragmentsmay be screened by nucleic acid hybridization to the labeled probe(Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. AndHogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Such aprocedure is presented by way of example in Section 7 infra. Those DNAfragments with substantial homology to the probe will hybridize. It isalso possible to identify the appropriate fragment by restriction enzymedigestion(s) and comparison of fragment sizes with those expectedaccording to a known restriction map if such is available. Furtherselection can be carried out on the basis of the properties of the gene.Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, kinase activity, inhibition ofcell proliferation activity, substrate binding activity, or antigenicproperties as known for lats. If an antibody to lats is available, thelats protein may be identified by binding of labeled antibody to theputatively lats synthesizing clones, in an ELISA (enzyme-linkedimmunosorbent assay)-type procedure.

The lats gene can also be identified by mRNA selection by nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization. SuchDNA fragments may represent available, purified lats DNA of anotherspecies (e.g., Drosophila, mouse, human). Immunoprecipitation analysisor functional assays (e.g., aggregation ability in vitro; binding toreceptor; see infra) of the in vitro translation products of theisolated products of the isolated mRNAs identifies the mRNA and,therefore, the complementary DNA fragments that contain the desiredsequences. In addition, specific mRNAs may be selected by adsorption ofpolysomes isolated from cells to immobilized antibodies specificallydirected against lats protein. A radiolabelled lats cDNA can besynthesized using the selected mRNA (from the adsorbed polysomes) as atemplate. The radiolabelled mRNA or cDNA may then be used as a probe toidentify the lats DNA fragments from among other genomic DNA fragments.

Alternatives to isolating the lats genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to the mRNA which encodes the latsprotein. For example, RNA for cDNA cloning of the lats gene can beisolated from cells which express lats. Other methods are possible andwithin the scope of the invention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas PBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene). The insertion into a cloning vector can, for example, beaccomplished by ligating the DNA fragment into a cloning vector whichhas complementary cohesive termini. However, if the complementaryrestriction sites used to fragment the DNA are not present in thecloning vector, the ends of the DNA molecules may be enzymaticallymodified. Alternatively, any site desired may be produced by ligatingnucleotide sequences (linkers) onto the DNA termini; these ligatedlinkers may comprise specific chemically synthesized oligonucleotidesencoding restriction endonuclease recognition sequences. In analternative method, the cleaved vector and lats gene may be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated lats gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

The lats sequences provided by the instant invention include thosenucleotide sequences encoding substantially the same amino acidsequences as found in native lats proteins, and those encoded amino acidsequences with functionally equivalent amino acids, as well as thoseencoding other lats derivatives or analogs, as described in Sections 5.6and 5.6.1 infra for lats derivatives and analogs.

5.2. EXPRESSION OF THE LATS GENES

The nucleotide sequence coding for a lats protein or a functionallyactive analog or fragment or other derivative thereof (see Section 5.6),can be inserted into an appropriate expression vector, i.e., a vectorwhich contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. The necessarytranscriptional and translational signals can also be supplied by thenative lats gene and/or its flanking regions. A variety of host-vectorsystems may be utilized to express the protein-coding sequence. Theseinclude but are not limited to mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophage,DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors varyin their strengths and specificities. Depending on the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements may be used. In specific embodiments, the humanlats gene is expressed, or a sequence encoding a functionally activeportion of human lats. In yet another embodiment, a fragment of latscomprising a domain of the lats protein is expressed.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequence encoding a lats protein or peptidefragment may be regulated by a second nucleic acid sequence so that thelats protein or peptide is expressed in a host transformed with therecombinant DNA molecule. For example, expression of a lats protein maybe controlled by any promoter/enhancer element known in the art.Promoters which may be used to control lats expression include, but arenot limited to, the SV40 early promoter region (Bernoist and Chambon,1981, Nature 290:304-310), the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 35 78:1441-1445), the regulatory sequencesof the metallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871),and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58;alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

In a specific embodiment, a vector is used that comprises a promoteroperably linked to a lats-encoding nucleic acid, one or more origins ofreplication, and, optionally, one or more selectable markers (e.g., anantibiotic resistance gene).

In a specific embodiment, an expression construct is made by subcloninga lats coding sequence into the EcoRI restriction site of each of thethree PGEX vectors (Glutathione S-Transferase expression vectors; Smithand Johnson, 1988, Gene 7:31-40). This allows for the expression of thelats protein product from the subclone in the correct reading frame.

Expression vectors containing lats gene inserts can be identified bythree general approaches: (a) nucleic acid hybridization, (b) presenceor absence of “marker” gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a lats gene insertedin an expression vector can be detected by nucleic acid hybridizationusing probes comprising sequences that are homologous to an insertedlats gene. In the second approach, the recombinant vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of a lats genein the vector. For example, if the lats gene is inserted within themarker gene sequence of the vector, recombinants containing the latsinsert can be identified by the absence of the marker gene function. Inthe third approach, recombinant expression vectors can be identified byassaying the lats product expressed by the recombinant. Such assays canbe based, for example, on the physical or functional properties of thelats protein in in vitro assay systems, e.g., kinase activity, bindingwith anti-lats antibody, inhibition of cell proliferation.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered lats protein may be controlled.Furthermore, different host cells have characteristic and specificmechanisms for the translational and post-translational processing andmodification (e.g., glycosylation, phosphorylation of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.For example, expression in a bacterial system can be used to produce anunglycosylated core protein product. Expression in yeast will produce aglycosylated product. Expression in mammalian cells can be used toensure “native” glycosylation of a heterologous protein. Furthermore,different vector/host expression systems may effect processing reactionsto different extents.

In other specific embodiments, the lats protein, fragment, analog, orderivative may be expressed as a fusion, or chimeric protein product(comprising the protein, fragment, analog, or derivative joined via apeptide bond to a heterologous protein sequence (of a differentprotein)). Such a chimeric product can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acidsequences to each other by methods known in the art, in the propercoding frame, and expressing the chimeric product by methods commonlyknown in the art. Alternatively, such a chimeric product may be made byprotein synthetic techniques, e.g., by use of a peptide synthesizer.

Both cDNA and genomic sequences can be cloned and expressed.

5.3. IDENTIFICATION AND PURIFICATION OF THE LATS GENE PRODUCTS

In particular aspects, the invention provides amino acid sequences oflats, preferably human lats, and fragments and derivatives thereof whichcomprise an antigenic determinant (i.e., can be recognized by anantibody) or which are otherwise functionally active, as well as nucleicacid sequences encoding the foregoing. “Functionally active” latsmaterial as used herein refers to that material displaying one or moreknown functional activities associated with a full-length (wild-type)lats protein, e.g., kinase activity, inhibition of cell proliferation,tumor inhibition, binding to an SH3-domain, binding to a lats substrateor lats binding partner, antigenicity (binding to an anti-latsantibody), immunogenicity, etc.

In specific embodiments, the invention provides fragments of a latsprotein consisting of at least 6 amino acids, 10 amino acids, 50 aminoacids, or of at least 75 amino acids. In other embodiments, the proteinscomprise or consist essentially of a lats carboxy (C)-terminal domain 3(LCD3), lats C-terminal domain 2 (LCD2), lats C-terminal domain 1(LCD1), kinase domain, kinase subdomains, lats flanking domain(amino-terminal to the kinase domain), lats split domain 1 (LSD1), latssplit domain 2 (LSD2), SH3-binding domain, and opa repeat domain (seeSection 8 infra), or any combination of the foregoing, of a latsprotein. Fragments, or proteins comprising fragments, lacking some orall of the foregoing regions of a lats protein are also provided.Nucleic acids encoding the foregoing are provided.

Once a recombinant which expresses the lats gene sequence is identified,the gene product can be analyzed. This is achieved by assays based onthe physical or functional properties of the product, includingradioactive labelling of the product followed by analysis by gelelectrophoresis, immunoassay, etc.

Once the lats protein is identified, it may be isolated and purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for the purification ofproteins. The functional properties may be evaluated using any suitableassay (see Section 5.7).

Alternatively, once a lats protein produced by a recombinant isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the chimeric gene contained in therecombinant. As a result, the protein can be synthesized by standardchemical methods known in the art (e.g., see Hunkapiller, M., et al.,1984, Nature 310:105-111).

In another alternate embodiment, native lats proteins can be purifiedfrom natural sources, by standard methods such as those described above(e.g., immunoaffinity purification).

In a specific embodiment of the present invention, such lats proteins,whether produced by recombinant DNA techniques or by chemical syntheticmethods or by purification of native proteins, include but are notlimited to those containing, as a primary amino acid sequence, all orpart of the amino acid sequence substantially as depicted in FIG. 9 (SEQID NO:4), as well as fragments and other derivatives, and analogsthereof, including proteins homologous thereto.

5.4. STRUCTURE OF THE LATS GENE AND PROTEIN

The structure of the lats gene and protein can be analyzed by variousmethods known in the art.

5.4.1. GENETIC ANALYSIS

The cloned DNA or cDNA corresponding to the lats gene can be analyzed bymethods including but not limited to Southern hybridization (Southern,E. M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (seee.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098),restriction endonuclease mapping (Maniatis, T., 1982, Molecular Cloning,A Laboratory, Cold Spring Harbor, N.Y.), and DNA sequence analysis.Polymerase chain reaction (PCR; U.S. Pat. Nos. 4,683,202, 4,683,195 and4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al.,1989, Science 243:217-220) followed by Southern hybridization with alats-specific probe can allow the detection of the lats gene in DNA fromvarious cell types. Methods of amplification other than PCR are commonlyknown and can also be employed. In one embodiment, Southernhybridization can be used to determine the genetic linkage of lats.Northern hybridization analysis can be used to determine the expressionof the lats gene. Various cell types, at various states of developmentor activity can be tested for lats expression. The stringency of thehybridization conditions for both Southern and Northern hybridizationcan be manipulated to ensure detection of nucleic acids with the desireddegree of relatedness to the specific lats probe used. Modifications ofthese methods and other methods commonly known in the art can be used.

Restriction endonuclease mapping can be used to roughly determine thegenetic structure of the lats gene. Restriction maps derived byrestriction endonuclease cleavage can be confirmed by DNA sequenceanalysis.

DNA sequence analysis can be performed by any techniques known in theart, including but not limited to the method of Maxam and Gilbert (1980,Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., etal., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNApolymerase (Tabor and Richardson, U.S. Pat. No. 4,795,699), or use of anautomated DNA sequenator (e.g., Applied Biosystems, Foster City,Calif.).

5.4.2. PROTEIN ANALYSIS

The amino acid sequence of the lats protein can be derived by deductionfrom the DNA sequence, or alternatively, by direct sequencing of theprotein, e.g., with an automated amino acid sequencer.

The lats protein sequence can be further characterized by ahydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identifythe hydrophobic and hydrophilic regions of the lats protein and thecorresponding regions of the gene sequence which encode such regions.

Secondary, structural analysis (Chou, P. and Fasman, G., 1974,Biochemistry 13:222) can also be done, to identify regions of lats thatassume specific secondary structures.

Manipulation, translation, and secondary structure prediction, openreading frame prediction and plotting, as well as determination ofsequence homologies, can also be accomplished using computer softwareprograms available in the art.

Other methods of structural analysis can also be employed. These includebut are not limited to X-ray 35 crystallography (Engstom, A., 1974,Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick, R. andZoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, inCurrent Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

5.5. GENERATION OF ANTIBODIES TO LATS PROTEINS AND DERIVATIVES THEREOF

According to the invention, lats protein, its fragments or otherderivatives, or analogs thereof, may be used as an immunogen to generateantibodies which immunospecifically bind such an immunogen. Suchantibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library. Ina specific embodiment, antibodies to a human lats protein are produced.In another embodiment, antibodies to a domain (e.g., the SH3-bindingdomain) of a lats protein are produced. In a specific embodiment,fragments of a lats protein identified as hydrophilic are used asimmunogens for antibody production.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a lats protein or derivative or analog. In aparticular embodiment, rabbit polyclonal antibodies to an epitope of alats protein encoded by a sequence of SEQ ID NOS:2, 4, 6 or 8, or asubsequence thereof, can be obtained. For the production of antibody,various host animals can be immunized by injection with the native latsprotein, or a synthetic version, or derivative (e.g., fragment) thereof,including but not limited to rabbits, mice, rats, etc. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a lats proteinsequence or analog thereof, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545).According to the invention, human antibodies may be used and can beobtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBVvirus in vitro (Cole et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, pp. 77-96). In fact, according to the invention,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing the genes from a mouse antibody moleculespecific for lats together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce lats-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., 1989, Science 246:1275-1281)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for lats proteins, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′) ₂ fragment which can be produced bypepsin digestion of the antibody molecule; the Fab′ fragments which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragment,the Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of a lats protein, one may assaygenerated hybridomas for a product which binds to a lats fragmentcontaining such domain. For selection of an antibody that specificallybinds a first lats homolog but which does not specifically bind adifferent lats homolog, one can select on the basis of positive bindingto the first lats homolog and a lack of binding to the second latshomolog.

Antibodies specific to a domain of a lats protein are also provided.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the lats protein sequencesof the invention, e.g., for imaging these proteins, measuring levelsthereof in appropriate physiological samples, in diagnostic methods,etc.

In another embodiment of the invention (see infra), anti-lats antibodiesand fragments thereof containing the binding domain are Therapeutics.

5.6. LATS PROTEINS, DERIVATIVES AND ANALOGS

The invention further relates to lats proteins, and derivatives(including but not limited to fragments) and analogs of lats proteins.Nucleic acids encoding lats protein derivatives and protein analogs arealso provided. In one embodiment, the lats proteins are encoded by thelats nucleic acids described in Section 5.1 supra. In particularaspects, the proteins, derivatives, or analogs are of lats proteins ofanimals, e.g., fly, frog, mouse, rat, pig, cow, dog, monkey, human, orof plants.

The production and use of derivatives and analogs related to lats arewithin the scope of the present invention. In a specific embodiment, thederivative or analog is functionally active, i.e., capable of exhibitingone or more functional activities associated with a full-length,wild-type lats protein. As one example, such derivatives or analogswhich have the desired immunogenicity or antigenicity can be used, forexample, in immunoassays, for immunization, for inhibition of latsactivity, etc. As another example, such derivatives or analogs whichhave the desired kinase activity, or which are phosphorylated ordephosphorylated, are provided. Derivatives or analogs that retain, oralternatively lack or inhibit, a desired lats property of interest(e.g., binding to an SH3-domain-containing protein or other lats bindingpartner, kinase activity, inhibition of cell proliferation, tumorinhibition), can be used as inducers, or inhibitors, respectively, ofsuch property and its physiological correlates. A specific embodimentrelates to a lats fragment that can be bound by an anti-lats antibody.Derivatives or analogs of lats can be tested for the desired activity byprocedures known in the art, including but not limited to the assaysdescribed in Sections 5.7 and 5.9.

In particular, lats derivatives can be made by altering lats sequencesby substitutions, additions or deletions that provide for functionallyequivalent molecules. Due to the degeneracy of nucleotide codingsequences, other DNA sequences which encode substantially the same aminoacid sequence as a lats gene may be used in the practice of the presentinvention. These include but are not limited to nucleotide sequencescomprising all or portions of lats genes which are altered by thesubstitution of different codons that encode a functionally equivalentamino acid residue within the sequence, thus producing a silent change.Likewise, the lats derivatives of the invention include, but are notlimited to, those containing, as a primary amino acid sequence, all orpart of the amino acid sequence of a lats protein including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be substituted by another amino acid of a similar polarity whichacts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In a specific embodiment of the invention, proteins consisting of orcomprising a fragment of a lats protein consisting of at least 10(continuous) amino acids of the lats protein is provided. In otherembodiments, the fragment consists of at least 20 or 50 amino acids ofthe lats protein. In specific embodiments, such fragments are not largerthan 35, 100 or 200 amino acids. Derivatives or analogs of lats includebut are not limited to those molecules comprising regions that aresubstantially homologous to lats or fragments thereof (e.g., in variousembodiments, at least 60% or 70% or 80% or 90% or 95% identity over anamino acid sequence of identical size or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art) or whose encoding nucleic acid is capable ofhybridizing to a coding lats sequence, under stringent, moderatelystringent, or nonstringent conditions.

The lats derivatives and analogs of the invention can be produced byvarious methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example,the cloned lats gene sequence can be modified by any of numerousstrategies known in the art (Maniatis, T., 1990, Molecular Cloning, ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). The sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative or analog of lats, care should be taken toensure that the modified gene remains within the same translationalreading frame as lats, uninterrupted by translational stop signals, inthe gene region where the desired lats activity is encoded.

Additionally, the lats-encoding nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson,C., et al., 1978, J. Biol. Chem 253:6551), use of TAB® linkers(Pharmacia), etc.

Manipulations of the lats sequence may also be made at the proteinlevel. Included within the scope of the invention are lats proteinfragments or other derivatives or analogs which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

In addition, analogs and derivatives of lats can be chemicallysynthesized. For example, a peptide corresponding to a portion of a latsprotein which comprises the desired domain (see Section 5.6.1), or whichmediates the desired activity in vitro, can be synthesized by use of apeptide synthesizer. Furthermore, if desired, nonclassical amino acidsor chemical amino acid analogs can be introduced as a substitution oraddition into the lats sequence. Non-classical amino acids include butare not limited to the D-isomers of the common amino acids, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, Cα-methyl aminoacids, Nα-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In a specific embodiment, the lats derivative is a chimeric, or fusion,protein comprising a lats protein or fragment thereof (preferablyconsisting of at least a domain or motif of the lats protein, or atleast 10 amino acids of the lats protein) joined at its amino- orcarboxy-terminus via a peptide bond to an amino acid sequence of adifferent protein. In one embodiment, such a chimeric protein isproduced by recombinant expression of a nucleic acid encoding theprotein (comprising a lats-coding sequence joined inframe to a codingsequence for a different protein). Such a chimeric product can be madeby ligating the appropriate nucleic acid sequences encoding the desiredamino acid sequences to each other by methods known in the art, in theproper coding frame, and expressing the chimeric product by methodscommonly known in the art. Alternatively, such a chimeric product may bemade by protein synthetic techniques, e.g., by use of a peptidesynthesizer. Chimeric genes comprising portions of lats fused to anyheterologous protein-encoding sequences may be constructed. A specificembodiment relates to a chimeric protein comprising a fragment of latsof at least six amino acids.

In another specific embodiment, the lats derivative is a moleculecomprising a region of homology with a lats protein. By way of example,in various embodiments, a first protein region can be considered“homologous” to a second protein region when the amino acid sequence ofthe first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or95% identical, when compared to any sequence in the second region of anequal number of amino acids as the number contained in the first regionor when compared to an aligned sequence of the second region that hasbeen aligned by a computer homology program known in the art. Forexample, a molecule can comprise one or more regions homologous to alats domain (see Section 5.6.1) or a portion thereof.

Other specific embodiments of derivatives and analogs are described inthe subsection below and examples sections infra.

5.6.1. DERIVATIVES OF LATS CONTAINING ONE OR MORE DOMAINS OF THE PROTEIN

In a specific embodiment, the invention relates to lats derivatives andanalogs, in particular lats fragments and derivatives of such fragments,that comprise, or alternatively consist of, one or more domains of alats protein, including but not limited to a lats C-terminal domain 3(LCD3), lats C-terminal domain 2 (LCD2), lats C-terminal domain 1(LCD1), kinase domain, kinase subdomains, lats flanking domain (LFD)(amino-terminal to the kinase domain), lats split domain 1 (LSD1), latssplit domain 2 (LSD2), SH3-binding domain, and opa repeat domain,functional (e.g., binding) fragments of any of the foregoing, or anycombination of the foregoing. In particular examples relating to thehuman, mouse and Drosophila lats proteins, such domains are identifiedin Examples Sections 6 and 8, and in FIGS. 6A, 6B, and 13.

A specific embodiment relates to molecules comprising specific fragmentsof lats that are those fragments in the respective lats protein mosthomologous to specific fragments of a human or mouse lats protein. Afragment comprising a domain of a lats homolog can be identified byprotein analysis methods as described in Sections 5.3.2 or 6.

In a specific embodiment, a lats protein, derivative or analog isprovided that has a kinase domain and has a phosphorylated serinesituated within 20 residues upstream of an Ala-Pro-Glu consensus insubdomain eight of its kinase domain. In another embodiment, a latsprotein derivative or analog is provided with a kinase domain and with adephosphorylated serine situated within 20 residues upstream of anAla-Pro-Glu consensus in subdomain eight of its kinase domain, or inwhich the serine situated within 20 residues upstream of that consensushas been deleted or substituted by another amino acid. In a specificembodiment, the invention provides various phosphorylated anddephosphorylated forms of the lats protein, derivative, or analog thatare active kinase forms. Both phosphorylation and dephosphorylation oflats at different residues could potentially activate lats. In anotherspecific embodiment, the invention provides various phosphorylated anddephosphorylated forms of the lats protein, derivative or analog thatare inactive kinase forms. Phosphorylation can be carried out by anymethods known in the art, e.g., by use of a kinase. Dephosphorylationcan be carried out by use of any methods known in the art, e.g., by useof a phosphatase.

Another specific embodiment relates to a derivative or analog of a latsprotein that is a dominant-active protein kinase. Such a derivative oranalog comprises a lats kinase domain that has been mutated so as to bedominantly active (exhibit constitutively active kinase activity). It isknown that acidic residues such as Glu and Asp sometimes mimic aphosphorylated residue, and changing the phosphorylatable Ser or Thrresidue in subdomain eight into a Glu or Asp residue has been previouslyused to produce constitutively active kinases (Mansour et al., 1994,Science 265:966-970). Thus, changing a serine or threonine residuesituated within 20 residues upstream of an Ala-Pro-Glu consensus insubdomain eight of a lats kinase domain into another residue (e.g., Glu,Asp) may be used to make a dominant-active lats protein kinase. Forexample, changing Ser9,4 in Drosophila lats, or changing Ser909 inh-lats, into a Glu residue could produce a dominant active lats kinase.

Another specific embodiment relates to a derivative or analog of latsthat is a dominant-negative protein kinase. Protein kinases can bemutated into dominant negative forms. Expression of a dominant negativeprotein kinase can suppress the activity of the wild-type form of thesame kinase. Dominant negative forms of protein kinases are oftenobtained by expressing an inactive form of a kinase (Milarski andSaltiel, 1994, J. Biol. Chem. 269(33):21239-21243) or by expressing anoncatalytic domain of a kinase (Lu and Means, 1994, EMBO J.12:2103-2113; Yarden et al., 1992, EMBO J. 11:2159-2166). Thus, a latsdominant-negative kinase can be obtained by mutating the kinase domainso as to be inactive (e.g., by deletion and/or point mutation). By wayof example, a lats derivative that is a dominant-negative kinase is alats protein that lacks a kinase domain but comprises one or more of theother domains of the lats protein; e.g., a lats protein derivativetruncated at about the beginning of the kinase domain (i.e., a latsfragment containing only sequences amino-terminal to the kinase domain).By way of another example, a lats derivative that is a dominant-negativekinase is a lats protein in which one of the residues conserved amongserine/threonine kinases (see Hanks et al., 1988, Science 241:42-52) ismutated (deleted or substituted by a different residue).

In another specific embodiment, a molecule is provided that comprisesone or more domains (or functional portion thereof) of a lats proteinbut that also lacks one or more domains (or functional portion thereof)of a lats protein. In particular examples, lats protein derivatives areprovided that lack an opa repeat domain. By way of another example, sucha protein may also lack all or a portion of the kinase domain, butretain at least the SH3-binding domain of a lats protein. In anotherembodiment, a molecule is provided that comprises one or more domains(or functional portion thereof) of a lats protein, and that has one ormore mutant (e.g., due to deletion or point mutation(s)) domains of alats protein (e.g., such that the mutant domain has decreased function).By way of example, the kinase domain may be mutant so as to havereduced, absent, or increased kinase activity.

5.7. ASSAYS OF THE LATS PROTEINS, DERIVATIVES AND ANALOGS

The functional activity of lats proteins, derivatives and analogs can beassayed by various methods.

For example, in one embodiment, where one is assaying for the ability tobind or compete with wild-type lats for binding to anti-lats antibody,various immunoassays known in the art can be used, including but notlimited to competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labelled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a lats-binding protein is identified, thebinding can be assayed, e.g., by means well-known in the art. In anotherembodiment, physiological correlates of lats binding to its substrates(signal transduction) can be assayed.

In another embodiment, kinase assays can be used to measure lats kinaseactivity. Such assays can be carried out by methods well known in theart. By way of example, a lats protein is contacted with a substrate(e.g., a known substrate of serine/threonine kinases) in the presence ofa ³²P-labeled phosphate donor, and any phosphorylation of the substrateis detected or measured.

In another embodiment, in insect or other model systems, genetic studiescan be done to study the phenotypic effect of a lats mutant that is aderivative or analog of wild-type lats (see Section 6, infra).

In addition, assays that can be used to detect or measure the ability toinhibit, or alternatively promote, cell proliferation are described inSection 5.9.

Other methods will be known to the skilled artisan and are within thescope of the invention.

5.8. THERAPEUTIC USES

The invention provides for treatment or prevention of various diseasesand disorders by administration of a therapeutic compound (termed herein“Therapeutic”). Such “Therapeutics” include but are not limited to: latsproteins and analogs and derivatives (including fragments) thereof(e.g., as described hereinabove); antibodies thereto (as describedhereinabove); nucleic acids encoding the lats proteins, analogs, orderivatives (e.g., as described hereinabove); lats antisense nucleicacids, and lats agonists and antagonists. Disorders involving celloverproliferation are treated or prevented by administration of aTherapeutic that promotes lats function. Disorders in which cellproliferation is deficient or is desired are treated or prevented byadministration of a Therapeutic that antagonizes (inhibits) latsfunction. The above is described in detail in the subsections below.

Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, a humanlats protein, derivative, or analog, or nucleic acid, or an antibody toa human lats protein, is therapeutically or prophylacticallyadministered to a human patient.

Additional descriptions and sources of Therapeutics that can be usedaccording to the invention are found in Sections 5.1 through 5.7 herein.

5.8.1. TREATMENT AND PREVENTION OF DISORDERS INVOLVING OVERPROLIFERATIONOF CELLS

Diseases and disorders involving cell overproliferation are treated orprevented by administration of a Therapeutic that promotes (i.e.,increases or supplies) lats function. Examples of such a Therapeuticinclude but are not limited to lats proteins, derivatives, or fragmentsthat are functionally active, particularly that are active in inhibitingcell proliferation (e.g., as demonstrated in in vitro assays or inanimal models or in Drosophila), and nucleic acids encoding a latsprotein or functionally active derivative or fragment thereof (e.g., foruse in gene therapy). Other Therapeutics that can be used, e.g., latsagonists, can be identified using in vitro assays or animal models, orassays in Drosophila, examples of which are described infra.

In specific embodiments, Therapeutics that promote lats function areadministered therapeutically (including prophylactically): (1) indiseases or disorders involving an absence or decreased (relative tonormal or desired) level of lats protein or function, for example, inpatients where lats protein is lacking, genetically defective,biologically inactive or underactive, or underexpressed; or (2) indiseases or disorders wherein in vitro (or in vivo) assays (see infra)indicate the utility of lats agonist administration. The absence ordecreased level in lats protein or function can be readily detected,e.g., by obtaining a patient tissue sample (e.g., from biopsy tissue)and assaying it in vitro for RNA or protein levels, structure and/oractivity of the expressed lats RNA or protein. Many methods standard inthe art can be thus employed, including but not limited to kinaseassays, immunoassays to detect and/or visualize lats protein (e.g.,Western blot, immunoprecipitation followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect lats expression by detecting and/orvisualizing lats mRNA (e.g., Northern assays, dot blots, in situhybridization, etc.), etc.

Diseases and disorders involving cell overproliferation that can betreated or prevented include but are not limited to malignancies,premalignant conditions (e.g., hyperplasia, metaplasia, dysplasia),benign tumors, hyperproliferative disorders, benign dysproliferativedisorders, etc. Examples of these are detailed below.

In a specific embodiment, the Therapeutic used, that promotes latsfunction, is a lats protein, derivative or analog comprising a latskinase domain (and optionally also a lats LFD, or the remainder of thelats sequence) in which a serine within 20 residues upstream of theAla-Pro-Glu consensus in subdomain eight of the kinase domain isphosphorylated or substituted by another residue (e.g., Glu, Asp).

In another specific embodiment, the Therapeutic used, that promotes latsfunction, is a derivative or analog comprising a kinase domain of a latsprotein that has been mutated so as to be dominantly active.

5.8.1.1. MALIGNANCIES

Malignancies and related disorders that can be treated or prevented byadministration of a Therapeutic that promotes lats function include butare not limited to those listed in Table 1 (for a review of suchdisorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. LippincottCo., Philadelphia):

TABLE 1 MALIGNANCIES AND RELATED DISORDERS Leukemia acute leukemia acutelymphocytic leukemia acute myelocytic leukemia myeloblasticpromyelocytic myelomonocytic monocytic erythroleukemia chronic leukemiachronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemiaPolycythemia vera Lymphoma Hodgkin's disease non-Hodgkin's diseaseMultiple myeloma Waldenström's macroglobulinemia Heavy chain diseaseSolid tumors sarcomas and carcinomas fibrosarcoma myxosarcomaliposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcomaendotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcomasynovioma mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcomacolon carcinoma pancreatic cancer breast cancer ovarian cancer prostatecancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweatgland carcinoma sebaceous gland carcinoma papillary carcinoma papillaryadenocarcinomas cystadenocarcinoma medullary carcinoma bronchogeniccarcinoma renal cell carcinoma hepatoma bile duct carcinomachoriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervicalcancer uterine cancer testicular tumor lung carcinoma small cell lungcarcinoma bladder carcinoma epithelial carcinoma glioma astrocytomamedulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastomaacoustic neuroma oligodendroglioma menangioma melanoma neuroblastomaretinoblastoma

In specific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders, aretreated or prevented in the bladder, breast, colon, lung, melanoma,pancreas, or uterus. In other specific embodiments, sarcoma, or leukemiais treated or prevented.

5.8.1.2. PREMALIGNANT CONDITIONS

The Therapeutics of the invention that promote lats activity can also beadministered to treat prenralignant conditions and to preventprogression to a neoplastic or malignant state, including but notlimited to those disorders listed in Table 1. Such prophylactic ortherapeutic use is indicated in conditions known or suspected ofpreceding progression to neoplasia or cancer, in particular, wherenon-neoplastic cell growth consisting of hyperplasia, metaplasia, ormost particularly, dysplasia has occurred (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W.B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a patient, can indicate the desirability ofprophylactic/therapeutic administration of a Therapeutic that promoteslats function. As mentioned supra, such characteristics of a transformedphenotype include morphology changes, looser substratum attachment, lossof contact inhibition, loss of anchorage dependence, protease release,increased sugar transport, decreased serum requirement, expression offetal antigens, disappearance of the 250,000 dalton cell surfaceprotein, etc. (see also id., at pp. 84-90 for characteristics associatedwith a transformed or malignant phenotype).

In a specific embodiment, leukoplakia, a benign-appearing hyperplasticor dysplastic lesion of the epithelium, or Bowen's disease, a carcinomain situ, are pre-neoplastic lesions indicative of the desirability ofprophylactic intervention.

In another embodiment, fibrocystic disease (cystic hyperplasia, mammarydysplasia, particularly adenosis (benign epithelial hyperplasia)) isindicative of the desirability of prophylactic intervention.

In other embodiments, a patient which exhibits one or more of thefollowing predisposing factors for malignancy is treated byadministration of an effective amount of a Therapeutic: a chromosomaltranslocation associated with a malignancy (e.g., the Philadelphiachromosome for chronic myelogenous leukemia, t(14;18) for follicularlymphoma, etc.), familial polyposis or Gardner's syndrome (possibleforerunners of colon cancer), benign monoclonal gammopathy (a possibleforerunner of multiple myeloma), and a first degree kinship with personshaving a cancer or precancerous disease showing a Mendelian (genetic)inheritance pattern (e.g., familial polyposis of the colon, Gardner'ssyndrome, hereditary exostosis, polyendocrine adenomatosis, medullarythyroid carcinoma with amyloid production and pheochromocytoma,Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen,retinoblastoma, carotid body tumor, cutaneous melanocarcinoma,intraocular melanocarcinoma, xeroderma pigmentosum, ataxiatelangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplasticanemia, and Bloom's syndrome; see Robbins and Angell, 1976, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.)

In another specific embodiment, a Therapeutic of the invention isadministered to a human patient to prevent progression to breast, colon,lung, pancreatic, or uterine cancer, or melanoma or sarcoma.

5.8.1.3. HYPERPROLIFERATIVE AND DYSPROLIFERATIVE DISORDERS

In another embodiment of the invention, a Therapeutic that promotes latsactivity is used to treat or prevent hyperproliferative or benigndysproliferative disorders. Specific embodiments are directed totreatment or prevention of cirrhosis of the liver (a condition in whichscarring has overtaken normal liver regeneration processes), treatmentof keloid (hypertrophic scar) formation (disfiguring of the skin inwhich the scarring process interferes with normal renewal), psoriasis (acommon skin condition characterized by excessive proliferation of theskin and delay in proper cell fate determination), benign tumors,fibrocystic conditions, and tissue hypertrophy (e.g., prostatichyperplasia).

5.8.1.4. GENE THERAPY

In a specific embodiment, nucleic acids comprising a sequence encoding alats protein or functional derivative thereof, are administered topromote lats function, by way of gene therapy. Gene therapy refers totherapy performed by the administration of a nucleic acid to a subject.In this embodiment of the invention, the nucleic acid produces itsencoded protein that mediates a therapeutic effect by promoting latsfunction.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11 (5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the Therapeutic comprises a lats nucleic acidthat is part of an expression vector that expresses a lats protein orfragment or chimeric protein thereof in a suitable host. In particular,such a nucleic acid has a promoter operably linked to the lats codingregion, said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the lats coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the lats nucleic acid (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra etal., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992(Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

In a specific embodiment, a viral vector that contains the lats nucleicacid is used. For example, a retroviral vector can be used (see Milleret al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. Thelats nucleic acid to be used in gene therapy is cloned into the vector,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; andMastrangeli et al., 1993, J. Clin. Invest. 91:225-234.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, alats nucleic acid is introduced into the cells such that it isexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention. Such stem cells include but are not limited to hematopoieticstem cells (HSC), stem cells of epithelial tissues such as the skin andthe lining of the gut, embryonic heart muscle cells, liver stem cells(PCT Publication WO 94/08598, dated Apr. 28, 1994), and neural stemcells (Stemple and Anderson, 1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,1980, Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) can alsobe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Techniques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allogeneic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see, e.g., Kodo et al.,1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79:3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Additional methods that can be adapted for use to deliver a nucleic acidencoding a lats protein or functional derivative thereof are describedin Section 5.8.2.2.2.

5.8.2. TREATMENT AND PREVENTION OF DISORDERS IN WHICH CELL PROLIFERATIONIS DESIRED

Diseases and disorders involving a deficiency in cell proliferation(growth) or in which cell proliferation is otherwise desirable fortreatment or prevention, are treated or prevented by administration of aTherapeutic that antagonizes (inhibits) lats function (in particular,lats-mediated inhibition of cell proliferation). Therapeutics that canbe used include but are not limited to anti-lats antibodies (andfragments and derivatives thereof containing the binding regionthereof), lats derivatives or analogs that are dominant-negativekinases, lats antisense nucleic acids, and lats nucleic acids that aredysfunctional (e.g., due to a heterologous (non-lats sequence) insertionwithin the lats coding sequence) that are used to “knockout” endogenouslats function by homologous recombination (see, e.g., Capecchi, 1989,Science 244:1288-1292). In a specific embodiment of the invention, anucleic acid containing a portion of a lats gene in which lats sequencesflank (are both 5′ and 3′ to) a different gene sequence, is used, as alats antagonist, to promote lats inactivation by homologousrecombination (see also Koller and Smithies, 1989, Proc. Natl. Acad.Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). OtherTherapeutics that inhibit lats function can be identified by use ofknown convenient in vitro assays, e.g., based on their ability toinhibit binding of lats to another protein (e.g., an SH3-domaincontaining protein), or inhibit any known lats function, as preferablyassayed in vitro or in cell culture, although genetic assays (e.g., inDrosophila) may also be employed. Preferably, suitable in vitro or invivo assays, are utilized to determine the effect of a specificTherapeutic and whether its administration is indicated for treatment ofthe affected tissue.

In specific embodiments, Therapeutics that inhibit lats function areadministered therapeutically (including prophylactically): (1) indiseases or disorders involving an increased (relative to normal ordesired) level of lats protein or function, for example, in patientswhere lats protein is overactive or overexpressed; or (2) in diseases ordisorders wherein in vitro (or in vivo) assays (see infra) indicate theutility of lats antagonist administration. The increased levels in latsprotein or function can be readily detected, e.g., by quantifyingprotein and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or protein levels,structure and/or activity of the expressed lats RNA or protein. Manymethods standard in the art can be thus employed, including but notlimited to kinase assays, immunoassays to detect and/or visualize latsprotein (e.g., Western blot, immunoprecipitation followed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry,etc.) and/or hybridization assays to detect lats expression by detectingand/or visualizing respectively lats mRNA (e.g., Northern assays, dotblots, in situ hybridization, etc.), etc.

Diseases and disorders involving a deficiency in cell proliferation orin which cell proliferation is desired for treatment or prevention, andthat can be treated or prevented by inhibiting lats function, includebut are not limited to degenerative disorders, growth deficiencies,hypoproliferative disorders, physical trauma, lesions, and wounds; forexample, to promote wound healing, or to promote regeneration indegenerated, lesioned or injured tissues, etc. In a specific embodiment,nervous system disorders are treated. In another specific embodiment, adisorder that is not of the nervous system is treated.

Lesions which may be treated according to the present invention includebut are not limited to the following lesions:

(i) traumatic lesions, including lesions caused by physical injury orassociated with surgery; (ii) ischemic lesions, in which a lack ofoxygen results in cell injury or death, e.g., myocardial or cerebralinfarction or ischemia, or spinal cord infarction or ischemia; (iii)malignant lesions, in which cells are destroyed or injured by malignanttissue; (iv) infectious lesions, in which tissue is destroyed or injuredas a result of infection, for example, by an abscess or associated withinfection by human immunodeficiency virus, herpes zoster, or herpessimplex virus or with Lyme disease, tuberculosis, syphilis; (v)degenerative lesions, in which tissue is destroyed or injured as aresult of a degenerative process, including but not limited to nervoussystem degeneration associated with Parkinson's disease, Alzheimer'sdisease; Huntington's chorea, or amyotrophic lateral sclerosis; (vi)lesions associated with nutritional diseases or disorders, in whichtissue is destroyed or injured by a nutritional disorder or disorder ofmetabolism including but not limited to, vitamin B12 deficiency, folicacid deficiency, Wernicke disease, tobacco-alcohol amblyopia,Marchiafava-Bignami disease (primary degeneration of the corpuscallosum), and alcoholic cerebellar degeneration; (vii) lesionsassociated with systemic diseases including but not limited to diabetesor systemic lupus erythematosus; (viii) lesions caused by toxicsubstances including alcohol, lead, or other toxins; and (ix)demyelinated lesions of the nervous system, in which a portion of thenervous system is destroyed or injured by a demyelinating diseaseincluding but not limited to multiple sclerosis, human immunodeficiencyvirus- associated myelopathy, transverse myelopathy or variousetiologies, progressive multifocal leukoencephalopathy, and centralpontine myelinolysis.

Nervous system lesions which may be treated in a patient (includinghuman and non-human mammalian patients) according to the inventioninclude but are not limited to the lesions of either the central(including spinal cord, brain) or peripheral nervous systems.

Therapeutics which are useful according to this embodiment of theinvention for treatment of a disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofcells (see also Section 5.9). For example, in a specific embodimentrelating to therapy of the nervous system, a Therapeutic which elicitsone of the following effects may be useful according to the invention:

(i) increased sprouting of neurons in culture or in vivo; (ii) increasedproduction of a neuron-associated molecule in culture or in vivo, e.g.,choline acetyltransferase or acetylcholinesterase with respect to motorneurons; or (iii) decreased symptoms of neuron dysfunction in vivo.

Such effects may be measured by any method known in the art. Inpreferred, non-limiting embodiments, increased sprouting of neurons maybe detected by methods set forth in Pestronk et al. (1980, Exp. Neurol.70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); andincreased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,depending on the molecule to be measured.

5.8.2.1. ANTISENSE REGULATION OF LATS EXPRESSION

In a specific embodiment, lats function is inhibited by use of latsantisense nucleic acids. The present invention provides the therapeuticor prophylactic use of nucleic acids of at least six nucleotides thatare antisense to a gene or cDNA encoding lats or a portion thereof. Alats “antisense” nucleic acid as used herein refers to a nucleic acidcapable of hybridizing to a portion of a lats RNA (preferably mRNA) byvirtue of some sequence complementarity. The antisense nucleic acid maybe complementary to a coding and/or noncoding region of a lats mRNA.Such antisense nucleic acids have utility as Therapeutics that inhibitslats function, and can be used in the treatment or prevention ofdisorders as described supra in Section 5.8.2 and its subsections.

The antisense nucleic acids of the invention can be oligonucleotidesthat are double-stranded or single-stranded, RNA or DNA or amodification or derivative thereof, which can be directly administeredto a cell, or which can be produced intracellularly by transcription ofexogenous, introduced sequences.

In a specific embodiment, the lats antisense nucleic acids provided bythe instant invention can be used to promote regeneration or woundhealing or to promote growth (larger size).

The invention further provides pharmaceutical compositions comprising aneffective amount of the lats antisense nucleic acids of the invention ina pharmaceutically acceptable carrier, as described infra.

In another embodiment, the invention is directed to methods forinhibiting the expression of a lats nucleic acid sequence in aprokaryotic or eukaryotic cell comprising providing the cell with aneffective amount of a composition comprising an lats antisense nucleicacid of the invention.

Lats antisense nucleic acids and their uses are described in detailbelow.

5.8.2.1.1. LATS ANTISENSE NUCLEIC ACIDS

The lats antisense nucleic acids are of at least six nucleotides and arepreferably oligonucleotides (ranging from 6 to about 50oligonucleotides). In specific aspects, the oligonucleotide is at least10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or atleast 200 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides, oragents facilitating transport across the cell membrane (see, e.g.,Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCTPublication No. WO 88/09810, published Dec. 15, 1988) or blood-brainbarrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25,1988), hybridization-triggered cleavage agents (see, e.g., Krol et al.,1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon,1988, Pharm. Res. 5:539-549).

In a preferred aspect of the invention, a lats antisense oligonucleotideis provided, preferably of single-stranded DNA. In a most preferredaspect, such an oligonucleotide comprises a sequence antisense to thesequence encoding an SH3 binding domain or a kinase domain of a latsprotein, most preferably, of a human lats protein. The oligonucleotidemay be modified at any position on its structure with substituentsgenerally known in the art.

The lats antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil,. 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the oligonucleotide is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641).

The oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

In a specific embodiment, the lats antisense oligonucleotide comprisescatalytic RNA, or a ribozyme (see, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). In another embodiment, the oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

In an alternative embodiment, the lats antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector can be introduced in vivo such that itis taken up by a cell, within which cell the vector or a portion thereofis transcribed, producing an antisense nucleic acid (RNA) of theinvention. Such a vector would contain a sequence encoding the latsantisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding thelats antisense RNA can be by any promoter known in the art to act inmammalian, preferably human, cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a lats gene,preferably a human lats gene. However, absolute complementarity,although preferred, is not required. A sequence “complementary to atleast a portion of an RNA,” as referred to herein, means a sequencehaving sufficient complementarity to be able to hybridize with the RNA,forming a stable duplex; in the case of double-stranded lats antisensenucleic acids, a single strand of the duplex DNA may thus be tested, ortriplex formation may be assayed. The ability to hybridize will dependon both the degree of complementarity and the length of the antisensenucleic acid. Generally, the longer the hybridizing nucleic acid, themore base mismatches with a lats RNA it may contain and still form astable duplex (or triplex , as the case may be). One skilled i n the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

5.8.2.1.2. THERAPEUTIC USE OF LATS ANTISENSE NUCLEIC ACIDS

The lats antisense nucleic acids can be used to treat (or prevent)disorders of a cell type that expresses, or preferably overexpresses,lats. In a specific embodiment, such a disorder is a growth deficiency.In a preferred embodiment, a single-stranded DNA antisense latsoligonucleotide is used.

Cell types which express or overexpress lats RNA can be identified byvarious methods known in the art. Such methods include but are notlimited to hybridization with a lats-specific nucleic acid (e.g. byNorthern hybridization, dot blot hybridization, in situ hybridization),observing the ability of RNA from the cell type to be translated invitro into lats, immunoassay, etc. In a preferred aspect, primary tissuefrom a patient can be assayed for lats expression prior to treatment,e.g., by immunocytochemistry or in situ hybridization.

Pharmaceutical compositions of the invention (see Section 5.10),comprising an effective amount of a lats antisense nucleic acid in apharmaceutically acceptable carrier, can be administered to a patienthaving a disease or disorder which is of a type that expresses oroverexpresses lats RNA or protein.

The amount of lats antisense nucleic acid which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. Where possible, it is desirable to determine theantisense cytotoxicity of the tumor type to be treated in vitro, andthen in useful animal model systems prior to testing and use in humans.

In a specific embodiment, pharmaceutical compositions comprising latsantisense nucleic acids are administered via liposomes, microparticles,or microcapsules. In various embodiments of the invention, it may beuseful to use such compositions to achieve sustained release of the latsantisense nucleic acids. In a specific embodiment, it may be desirableto utilize liposomes targeted via antibodies to specific identifiabletumor antigens (Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).

Additional methods that can be adapted for use to deliver a latsantisense nucleic acid are described in Section 5.8.1.4.

5.9. DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC UTILITY

The Therapeutics of the invention are preferably tested in vitro, andthen in vivo for the desired therapeutic or prophylactic activity, priorto use in humans. For example, In vitro assays which can be used todetermine whether administration of a specific Therapeutic is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered aTherapeutic, and the effect of such Therapeutic upon the tissue sampleis observed. In one embodiment, where the patient has a malignancy, asample of cells from such malignancy is plated out or grown in culture,and the cells are then exposed to a Therapeutic. A Therapeutic whichinhibits survival or growth of the malignant cells is selected fortherapeutic use in vivo. Many assays standard in the art can be used toassess such survival and/or growth; for example, cell proliferation canbe assayed by measuring ³H-thymidine incorporation, by direct cellcount, by detecting changes in transcriptional activity of known genessuch as proto-oncogenes (e.g., fos, myc) or cell cycle markers; cellviability can be assessed by trypan blue staining, differentiation canbe assessed visually based on changes in morphology, etc.

In another embodiment, a Therapeutic is indicated for use which exhibitsthe desired effect, inhibition or promotion of cell growth, upon apatient cell sample from tissue having or suspected of having a hyper-or hypoproliferative disorder, respectively. Such hyper- orhypoproliferative disorders include but are not limited to thosedescribed in Sections 5.8.1 through 5.8.3 infra.

In another specific embodiment, a Therapeutic is indicated for use intreating cell injury or a degenerative disorder (see Section 5.8.2)which exhibits in vitro promotion of growth/proliferation of cells ofthe affected patient type. Regarding nervous system disorders, see alsoSection 5.8.2.1 for assays that can be used.

In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if a Therapeutic has a desired effect upon such cell types.

In another embodiment, cells of a patient tissue sample suspected ofbeing pre-neoplastic are similarly plated out or grown in vitro, andexposed to a Therapeutic. The Therapeutic which results in a cellphenotype that is more normal (i.e., less representative of apre-neoplastic state, neoplastic state, malignant state, or transformedphenotype) is selected for therapeutic use. Many assays standard in theart can be used to assess whether a pre-neoplastic state, neoplasticstate, or a transformed or malignant phenotype, is present. For example,characteristics associated with a transformed phenotype (a set of invitro characteristics associated with a tumorigenic ability in vivo)include a more rounded cell morphology, looser substratum attachment,loss of contact inhibition, loss of anchorage dependence, release ofproteases such as plasminogen activator, increased sugar transport,decreased serum requirement, expression of fetal antigens, disappearanceof the 250,000 dalton surface protein, etc. (see Luria et al., 1978,General Virology, 3d Ed., John Wiley & Sons, New York pp. 436-446).

In other specific embodiments, the in vitro assays described supra canbe carried out using a cell line, rather than a cell sample derived fromthe specific patient to be treated, in which the cell line is derivedfrom or displays characteristic(s) associated with the malignant,neoplastic or pre-neoplastic disorder desired to be treated orprevented, or is derived from the cell type upon which an effect isdesired, according to the present invention.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to rats,mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, priorto administration to humans, any animal model system known in the artmay be used.

5.10. THERAPEUTIC/PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a Therapeutic ofthe invention. In a preferred aspect, the Therapeutic is substantiallypurified. The subject is preferably an animal, including but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andis preferably a mammal, and most preferably human. In a specificembodiment, a non-human mammal is the subject.

Formulations and methods of administration that can be employed when theTherapeutic comprises a nucleic acid are described in Sections 5.8.1.4and 5.8.2.2 above; additional appropriate formulations and routes ofadministration can be selected from among those described hereinbelow.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeuticnucleic acid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In another embodiment, the Therapeutic can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the Therapeutic can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the Therapeutic is a nucleic acidencoding a protein Therapeutic, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeuticcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine,procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.11. ADDITIONAL USE OF INHIBITION OF LATS FUNCTION TO PROMOTE INCREASEDGROWTH

Inhibition of lats function (e.g., by administering a compound thatinhibits lats function as described in Sections 5.8.2 through 5.8.2.1.2above), has utility that is not limited to therapeutic or prophylacticapplications. For example, lats function can be inhibited in order toincrease growth of animals (e.g., cows, horses, pigs, goats, deer,chickens) and plants (particularly edible plants, e.g., tomatoes,melons, lettuce, carrots, potatoes, and other vegetables), particularlythose that are food or material sources. For example, antisenseinhibition (preferably where the lats antisense nucleic acid is underthe control of a tissue-specific promoter) can be used in plants oranimals to increase growth where desired (e.g., in the fruit or muscle).For example, a lats antisense nucleic acid under the control of atemperature-sensitive promoter can be administered to a plant or animal,and the desired portion of the (or the entire) plant or animal can besubjected to heat in order to induce antisense nucleic acid production,resulting lats inhibition, and resulting cell proliferation. In otherembodiments, chemical mutagenesis, or homologous recombination with aninsertionally inactivated lats gene (see Capecchi, 1989, Science244:1288-1292 and Section 5.14 infra) can be carried out to reduce ordestroy endogenous lats function, in order to achieve increased growth.Suitable methods, modes of administration and compositions, that can beused to inhibit lats function are described in Sections 5.8.2 through5.8.2.1.2, above. Methods to make plants recombinant are commonly knownin the art and can be used. Regarding methods of plant transformation(e.g., for transformation with a lats antisense nucleic acid or with asequence encoding a lats derivative that is a dominant-negative kinase),see e.g., Valvekens et al., 1988, Proc. Natl. Acad. Sci. USA85:5536-5540. Regarding methods of targeted gene inactivation in plants(e.g., to inactivate lats), see e.g., Miao and Lam, 1995, The Plant J.7:359-365.

Inhibition of lats function can also have uses in vitro, e.g., to expandcells in vitro, including but not limited to stem cells, progenitorcells, muscle cells, fibroblasts, liver cells, etc., e.g., to growcells/tissue in vitro prior to administration to a patient (preferably apatient from which the cells were derived), etc.

5.12. DIAGNOSIS AND SCREENING

Lats proteins, analogues, derivatives, and subsequences thereof, latsnucleic acids (and sequences complementary thereto), anti-latsantibodies, have uses in diagnostics. Such molecules can be used inassays, such as immunoassays, to detect, prognoses, diagnose, or monitorvarious conditions, diseases, and disorders affecting lats expression,or monitor the treatment thereof. In particular, such an immunoassay iscarried out by a method comprising contacting a sample derived from apatient with an anti-lats antibody under conditions such thatimmunospecific binding can occur, and detecting or measuring the amountof any immunospecific binding by the antibody. In a specific aspect,such binding of antibody, in tissue sections, can be used to detectaberrant lats localization or aberrant (e.g., low or absent) levels oflats. In a specific embodiment, antibody to lats can be used to assay ina patient tissue or serum sample for the presence of lats where anaberrant level of lats is an indication of a diseased condition. By“aberrant levels,” is meant increased or decreased levels relative tothat present, or a standard level representing that present, in ananalogous sample from a portion of the body or from a subject not havingthe disorder.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew.

Lats genes and related nucleic acid sequences and subsequences,including complementary sequences, can also be used in hybridizationassays. Lats nucleic acid sequences, or subsequences thereof comprisingabout at least 8 nucleotides, can be used as hybridization probes.Hybridization assays can be used to detect, prognose, diagnose, ormonitor conditions, disorders, or disease states associated withaberrant changes in lats expression and/or activity as described supra.In particular, such a hybridization assay is carried out by a methodcomprising contacting a sample containing nucleic acid with a nucleicacid probe capable of hybridizing to lats DNA or RNA, under conditionssuch that hybridization can occur, and detecting or measuring anyresulting hybridization.

In specific embodiments, diseases and disorders involvingoverproliferation of cells can be diagnosed, or their suspected presencecan be screened for, or a predisposition to develop such disorders canbe detected, by detecting decreased levels of lats protein, lats RNA, orlats functional activity (e.g., kinase activity, SH3 domain-bindingactivity, etc.), or by detecting mutations in lats RNA, DNA or protein(e.g., translocations in lats nucleic acids, truncations in the latsgene or protein, changes in nucleotide or amino acid sequence relativeto wild-type lats) that cause decreased expression or activity of lats.Such diseases and disorders include but are not limited to thosedescribed in Section 5.8.1 and its subsections. By way of example,levels of lats protein can be detected by immunoassay, levels of latsRNA can be detected by hybridization assays (e.g., Northern blots, dotblots), lats kinase activity can be measured by kinase assays commonlyknown in the art, lats binding to an SH3 domain-containing protein canbe done by binding assays commonly known in the art, translocations andpoint mutations in lats nucleic acids can be detected by Southernblotting, RFLP analysis, PCR using primers that preferably generate afragment spanning at least most of the lats gene, sequencing of the latsgenomic DNA or cDNA obtained from the patient, etc.

In a preferred embodiment, levels of lats MRNA or protein in a patientsample are detected or measured, in which decreased levels indicate thatthe subject has, or has a predisposition to developing, a malignancy orhyperproliferative disorder; in which the decreased levels are relativeto the levels present in an analogous sample from a portion of the bodyor from a subject not having the malignancy or hyperproliferativedisorder, as the case may be.

In another specific embodiment, diseases and disorders involving adeficiency in cell proliferation or in which cell proliferation isdesirable for treatment, are diagnosed, or their suspected presence canbe screened for, or a predisposition to develop such disorders can bedetected, by detecting increased levels of lats protein, lats RNA, orlats functional activity (e.g., kinase activity, SH3 domain bindingactivity, etc.), or by detecting mutations in lats RNA, DNA or protein(e.g., translocations in lats nucleic acids, truncations in the gene orprotein, changes in nucleotide or amino acid sequence relative towild-type lats) that cause increased expression or activity of lats.Such diseases and disorders include but are not limited to thosedescribed in Section 5.8.2 and its subsections. By way of example,levels of lats protein, levels of lats RNA, lats kinase activity, latsbinding activity, and the presence of translocations or point mutationscan be determined as described above.

In a specific embodiment, levels of lats mRNA or protein in a patientsample are detected or measured, in which increased levels indicate thatthe subject has, or has a predisposition to developing, a growthdeficiency or degenerative or hypoproliferative disorder; in which theincreased levels are relative to the levels present in an analogoussample from a portion of the body or from a subject not having thegrowth deficiency, degenerative, or hypoproliferative disorder, as thecase may be.

Kits for diagnostic use are also provided, that comprise in one or morecontainers an anti-lats antibody, and, optionally, a labeled bindingpartner to the antibody.

Alternatively, the anti-lats antibody can be labeled (with a detectablemarker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactivemoiety). A kit is also provided that comprises in one or more containersa nucleic acid probe capable of hybridizing to lats RNA. In a specificembodiment, a kit can comprise in one or more containers a pair ofprimers (e.g., each in the size range of 6-30 nucleotides) that arecapable of priming amplification [e.g., by polymerase chain reaction(see e.g., Innis et al., 1990, PCR Protocols, Academic Press, Inc., SanDiego, Calif.), ligase chain reaction (see EP 320,308) use of Qβreplicase, cyclic probe reaction, or other methods known in the art]under appropriate reaction conditions of at least a portion of a latsnucleic acid. A kit can optionally further comprise in a container apredetermined amount of a purified lats protein or nucleic acid, e.g.,for use as a standard or control.

5.13. SCREENING FOR LATS AGONISTS AND ANTAGONISTS

Lats nucleic acids, proteins, and derivatives also have uses inscreening assays to detect molecules that specifically bind to latsnucleic acids, proteins, or derivatives and thus have potential use asagonists or antagonists of lats, in particular, molecules that thusaffect cell proliferation. In a preferred embodiment, such assays areperformed to screen for molecules with potential utility as anti-cancerdrugs or lead compounds for drug development. The invention thusprovides assays to detect molecules that specifically bind to latsnucleic acids, proteins, or derivatives. For example, recombinant cellsexpressing lats nucleic acids can be used to recombinantly produce latsproteins in these assays, to screen for molecules that bind to a latsprotein. Molecules (e.g., putative binding partners of lats) arecontacted with the lats protein (or fragment thereof) under conditionsconducive to binding, and then molecules that specifically bind to thelats protein are identified. Similar methods can be used to screen formolecules that bind to lats derivatives or nucleic acids. Methods thatcan be used to carry out the foregoing are commonly known in the art.

By way of example, diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries can be screened for molecules thatspecifically bind to lats. Many libraries are known in the art that canbe used, e.g., chemically synthesized libraries, recombinant (e.g.,phage display libraries), and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor etal., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86;Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb etal., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al.,1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad.Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described 30 in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all toLadner et al.; Rebar and Pabo, 1993, Science 263:671-673; and PCTPublication No. WO 94/18318.

In a specific embodiment, screening can be carried out by contacting thelibrary members with a lats protein (or nucleic acid or derivative)immobilized on a solid phase and harvesting those library members thatbind to the protein (or nucleic acid or derivative). Examples of suchscreening methods, termed “panning” techniques are described by way ofexample in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al.,1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and inreferences cited hereinabove.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien,etal., .1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to a lats protein orderivative.

In addition, Drosophila can be used as a model system in order to detectgenes that phenotypically interact with lats. For example,overexpression of lats in Drosophila eye leads to a smaller and roughereye. Mutagenesis of the fly genome can be performed, followed byselecting flies in which the mutagenesis has resulted in suppression orenhancement of the small rough eye phenotype; the mutated genes in suchflies are likely to encode proteins that interact/bind with lats.

5.14. ANIMAL MODELS

The invention also provides animal models.

In one embodiment, animal models for diseases and disorders involvingcell overproliferation (e.g., as described in Section 5.8.1) areprovided. Such an animal can be initially produced by promotinghomologous recombination between a lats gene in its chromosome and anexogenous lats gene that has been rendered biologically inactive(preferably by insertion of a heterologous sequence, e.g., an antibioticresistance gene). In a preferred aspect, this homologous recombinationis carried out by transforming embryo-derived stem (ES) cells with avector containing the insertionally inactivated lats gene, such thathomologous recombination occurs, followed by injecting the ES cells intoa blastocyst, and implanting the blastocyst into a foster mother,followed by the birth of the chimeric animal (“knockout animal”) inwhich a lats gene has been inactivated (see Capecchi, 1989, Science244:1288-1292). The chimeric animal can be bred to produce additionalknockout animals. Such animals can be mice, hamsters, sheep, pigs,cattle, etc., and are preferably non-human mammals. In a specificembodiment, a knockout mouse is produced.

Such knockout animals are expected to develop or be predisposed todeveloping diseases or disorders involving cell overproliferation (e.g.,malignancy) and thus can have use as animal models of such diseases anddisorders, e.g., to screen for or test molecules (e.g., potentialanti-cancer therapeutics) for the ability to inhibit overproliferation(e.g., tumor formation) and thus treat or prevent such diseases ordisorders.

In a different embodiment of the invention, transgenic animals that haveincorporated and express a functional lats gene have use as animalmodels of diseases and disorders involving deficiencies in cellproliferation or in which cell proliferation is desired. Such animalscan be used to screen for or test molecules for the ability to promoteproliferation and thus treat or prevent such diseases and disorders.

5.15. METHODS OF IDENTIFYING TUMOR SUPPRESSOR GENES AND OTHER GENES WITHIDENTIFIABLE PHENOTYPES

The invention also provides methods of identifying a tumor suppressorgene (or potential tumor suppressor gene) comprising identifying anoverproliferation phenotype in a genetic mosaic, and isolating a genethat is mutated in cells exhibiting the overproliferation phenotype. Thegenetic mosaic is achieved by induction of somatic cells in an animalthat is heterozygous for an induced mutation to become homozygous forthe mutation, at any desired developmental stage. The mutation can beinduced by any known method, e.g., X-ray exposure or chemical mutationexposure or insertion of a transposable element (e.g., P-element). Agenetic mosaic is produced by induction of homozygosity by mitoticrecombination between homologous arms of both parental chromosomes,which is achieved using a site-specific recombination system [a sequencecapable of expressing a site-specific recombinase; and its target sites(sequences at which the recombinase promotes recombination)], that havebeen inserted in the homozygous arms of both parental chromosomes. Thetarget sites are preferably inserted close to the centromere on eachchromosome arm (the closer to the centromere, the more preferred), sothat mitotic recombination events will result in cells being homozygousfor the mutation located on the chromosome arm distal to the insertionof the target site. For example, an FLP recombinase can be used with FRTtarget sites; Cre recombinase can be used with lox target sites. Therecombinase coding sequence, used to express recombinase, preferably,but need not be, intrachromosomally situated. For at least onechromosome, the target sites are intrachromosomally inserted on thehomologous arms of both parental (maternal and paternal) chromosomes.

The genetic mosaic can be an animal, e.g., mouse, hamster, sheep, pig,cow, Drosophila, etc., and is preferably a non-human mammal.

In a specific embodiment relating to the production of a non-humanmammal that is a genetic mosaic, a recombinase target site is introducedonto one arm of a chromosome in an embryo-derived stem cell (ES). Thetarget site can be introduced into the cell by homologous recombination(by use of flanking sequences from the desired site of intrachromosomalintegration) or by random integration resulting from cell transformation(e.g., by transfection, electroporation), etc. This ES is then injectedinto a blastocyst, the blastocyst is implanted into a foster mother,followed by birth of the recombinant animal. This mammal is bred to awild-type female, to produce siblings. Siblings carrying the target siteinsertion are mated, and offspring carrying the target site on thehomologous arms of both parental chromosomes are isolated (“the targetstrain”). A target strain member is then mutagenized and mated with anon-mutagenized target strain member of the opposite sex (preferablyalso carrying a recombinant nucleic acid encoding and capable ofexpressing a recombinase that promotes recombination at the targetsites), to obtain a target strain member that is heterozygous for themutation. Provision of the recombinase (by expression) in mitoticallyactive cells of a developing animal or an adult animal promotes mitoticrecombination between the homologous arms of the parental chromosomes,resulting in a cell that is homozygous for the mutation. Cells thatdisplay a mutant phenotype by virtue of their being homozygous for themutation are then detected, and the mutant gene can be geneticallymapped by any known method, and can be isolated.

In a Drosophila animal, a site-specific recombination system can beintroduced by use of P-element-mediated insertions.

In one embodiment, target sites are introduced onto homologous arms ofboth of a set of parental chromosomes, for one chromosome. In anotherembodiment, target sites are introduced onto homologous arms of both ofa set of parental chromosomes, for a plurality of chromosomes.

The recombinase can be under the control of a constitutive (e.g.,phosphorylated kinase promoter) or inducible (e.g., heat shock promoter)or tissue-specific promoter. The recombinase can be expressed episomally(e.g., from a plasmid) or chromosomally. Once the recombination systemis introduced into the animal, genetic mosaicism is produced by theactivity of the recombinase (which promotes recombination at the targetsites).

In a specific embodiment, an animal is used that contains a recombinantnucleic acid encoding an FLP recombinase (Broach and Hicks, 1980, Cell21:501-508) such that it is expressible by a cell of the animal, andintrachromosomal insertions of an FRT site on the homologous arms ofboth parental chromosomes; and genetic mosaicism is produced by inducingmitotic recombination between the FRT sites on the homologous chromosomearms after FLP recombinase expression (e.g., by heat shock, whenexpression of the FLP recombinase is under the control of a heat shockpromoter).

In another specific embodiment, an animal is used that contains arecombinant nucleic acid encoding a Cre recombinase (Sauer andHenderson, 1988, Proc. Natl. Acad. Sci. USA 85:5166-5170) such that itis expressible by a cell of the animal, and intrachromosomal insertionsof a lox site on homologous arms of both parental chromosomes; andgenetic mosaicism is produced by inducing mitotic recombination betweenthe lox sites on the homologous chromosome arms after Cre recombinaseexpression.

The animal may optionally further comprise intrachromosomal insertionsof marker genes (comprising a sequence encoding a protein containing areporter group such as an epitope tag), to facilitate confirmationand/or monitoring of recombination events. For example, in a non-humanmammal, a marker gene (e.g., lacZ) operably linked to a constitutivepromoter can be inserted, on the same chromosome arm as that carryingthe target site and the induced mutation.

In a specific embodiment, the overproliferation phenotype is theformation of overproliferated outgrowth tissue in anon-position-dependent fashion. In another specific embodiment, theoverproliferation phenotype is the formation of a normal structure oflarger than normal size.

The above-described genetic mosaics have uses not only in identifyingtumor suppressor genes, but, more generally, in identifying genes withan identifiable phenotype, i.e., those genes which in mutated form causean observable mutant phenotype to be displayed in the genetic mosaic.

In another embodiment, the invention provides a method of identifyinggenes with an observable mutant phenotype by use of human (or otheranimal) tissue culture cells that have incorporated a site-specificrecombination system such as described above. The site-specificrecombination system can be introduced by methods such as describedabove, so as to introduce a recombinant source of recombinase and effectintrachromosomal insertions of the recombinase target sites on thehomologous arms of both of a set of parental chromosomes, for one ormore chromosomes. In a preferred aspect relating to this use of culturecells, the recombinase target sites are ligated to a selectable marker(e.g., an antibiotic resistance gene), and cells are obtained that havethe target sites on each of the homologous chromosome arms, by selectingunder selection conditions of relatively high stringency (e.g., byincreasing the antibiotic concentration in the cell medium), As with theuse of genetic mosaics as described above, once mitotic recombination isinduced between the target sites on the homologous chromosome arms, onethen identifies-cells displaying a mutant phenotype, and recovers a genemutated in cells exhibiting the mutant phenotype. For example, apotential tumor suppressor gene can be identified by isolating a genethat is mutated in cultured cells exhibiting a transformed phenotype.

6. IDENTIFYING TUMOR SUPPRESSORS IN GENETIC MOSAICS: THE DROSOPHILA LATSGENE ENCODES A PUTATIVE PROTEIN KINASE

We have identified recessive overproliferation mutations by screeningand examining clones of mutant cells in genetic mosaics of the fruitflyDrosophila melanogaster (FIG. 1A). Flies that carry small groups ofsomatic cells mutated for negative regulators of cell proliferation ortumor suppressors are viable, yet the overproliferated mutant tissuescan be readily identifiable.

One way to generate mosaic animals is to induce mitotic recombination indeveloping heterozygous individuals (FIG. 1B). Recently, it was foundthat the site-specific recombination system from yeast, the FLPrecombinase and its target site FRT, can be used to induce highfrequency of mitotic recombination in Drosophila (Golic and Lindquist,1989, Cell 59:499-509; Golic, 1991, Science 252:958-961). To produce andanalyze genetic mosaics, a series of special Drosophila strains wereconstructed, containing the FLP/FRT recombination system on geneticallymarked chromosomes (Xu and Rubin, 1993, Development 117:1223-1237).Using these strains, high frequencies of mosaicism can be produced formore than 95% of the Drosophila genes. We have used these strains toidentify overproliferation mutations in mosaic animals.

Our results show that screening for overproliferation mutations inmosaic animals is a powerful way to identify negative regulators of cellproliferation and potential tumor suppressor genes. One of theidentified genes, large tumor suppressor (lats), has been cloned, andencodes a predicted novel protein kinase. Mutations in lats causedramatic overproliferation phenotypes and various developmental defectsin both mosaic animals and homozygous mutants.

6.1. MATERIALS AND METHODS

Genetics

Fly stocks and crosses were grown on standard medium at 25° C. unlessotherwise indicated. The F1 mosaic screens were modified from the onedescribed in Xu and Rubin (1993, Development 117:1223-1237) and in Xuand Harrison (1994, Methods in Cell Biology 44:655-682). Briefly, the F1mosaic individuals were produced from three crosses: Mutagenized y whsFLP1; P[ry⁺; hs-neo; FRT]40A males were mated to the y w hsFLP1;P[ry⁺; y⁺]25F, P[mini-w⁺; hs-NM]31E, P[ry⁺; hs-neo; FRT]40A females.Mutagenized y w hsFLP1; P[ry⁺; hs-neo; FRT]42D males were mated to the yw hsFLP1; P[ry⁺; hs-neo; FRT]42D, P[ry⁺; y⁺]44B, P[mini-w⁺;hs-NM]46F/CyO females. Finally, mutagenized y w hsFLP1; P[ry⁺; hs-neo;FRT]82B males were mated to the y w hsFLP1; P[ry⁺; hs-neo; FRT]82B,P[mini-w⁺; hs-πM]87E, Sb^(63b), P[ry⁺; y⁺]96E females. The male parentswere irradiated with X-rays (4000 r) and were removed from the crossesafter four days of mating. The eggs from the crosses were collected forevery 12 hours and aged for another 30 hours before being incubated in a38° C. water bath for 60 minutes. The F₁ animals were then returned tonormal culture conditions until eclosion. About 25,000 F₁ adults fromthese crosses were examined. Each P-induced lethal mutation wasrecombined onto one of the FRT-carrying arms using the neo^(R) and w⁻double selection as described in Xu and Harrison (1994, Methods in CellBiology 44:655-682) before examining its clonal phenotype.

The lats^(x1) mutation was meiotically mapped to the right of claret. Itwas further localized to the 100A1-5 region since it complemented Df(3R)tll^(e)(100A2-5; 100C2-3) and failed to complementDf(3R)tll^(pgx)(100A1-2; 100B4-5) and Df(3R) tll²⁰(100A1-3; 100B1-2). Asaturation genetic screen had previously been performed for thisinterval, and three lethal complementation groups, 1(3)100Aa, 1(3)100Aband the zfh-1, were isolated (Lai et al., 1993, Proc. Natl. Acad. Sci.USA 90:4122-4126). The lats^(x1) mutation failed to complement theEMS-induced mutations in 1(3)100Aa (lats^(a1-a15)), but complementmutations in 1(3)100Ab and zfh-1. The clonal phenotypes were examinedfor lats^(x1, P1, a1, a2, a6 and a10) induced either with theFLP/FRT-marker system or X-ray irradiation.

The lats^(P1) allele was recovered from a mosaic male produced from thecross of y w hsFLP1; P[ry⁺; hs-neo; FRT]82B x y w P[lacZ; w⁺]5; P[ry⁺;hs-neo; FRT]82B/delta2-3, Sb. The mutant chromosome was cleaned upbefore performing complementation tests and an excision screen(Robertson et al., 1988, Genetics 118:461-470). Two hundred and fifteenexcision lines were established that had lost the w⁺gene in the P[lacZ;w⁺] element (Bier et al., 1989, Genes Dev. 3:1273-1287). In about 50% ofthese lines, the pupal lethality had been reverted completely to wildtype, indicating the mutant phenotype is caused by the P-elementinsertion. Five lines were found to cause lethality at late embryonicand/or early first instar larval stages. The remaining lines were foundto cause lethality at larval and pupal stages or to produce viablemutant animals. All of these mutant excision lines (except one which islocated outside the 100A1-5 region) failed to complement lats^(x1)andlats^(P1), but do complement mutations in the zfh-1 and 1 (3)100Ab loci.

The insert in lats cDNA A2 was cloned into the pCaSpeR-hs vector(Thummel and Pirrotta, 1992, Drosophila Inform. Service 71:150) for germline transformation. Three of the transformed lines were tested and wereable to rescue the lethality of the lats^(a1)/lats^(x1), lats^(P1) andlats^(e26-1) animals after one hour heat shock for every 24 hours duringlarval and pupal development.

Histology

Fixation and sectioning (2 mm) of adult Drosophila tissues wereperformed as described (Tomlinson and Ready, 1987, Dev. Biol.123:264-275). Scanning electron microscopy was performed as described(Xu and Artavanis-Tsakonas, 1990, Genetics 126:665-677).

Nucleic Acid Manipulation

A P1 genomic clone (DS02640) mapped in the 100A1-7 region was obtainedfrom the Berkeley Drosophila Genome Center (personal communication;Hartl et al., 1994, Proc. Natl. Acad. Sci. USA 91:6824-6829). DNAfragments from this P1 clone and genomic DNA obtained by plasmid rescuefrom the lats^(P1) mutant (Bier et al., 1989, Genes Dev. 3:1273-1287)were used to isolate several overlapping cosmids including CLT-52 fromthe genomic library prepared by J. Tamkun. Genomic DNA from +7.5 (BglII)to −4.2 (EcoRI; FIG. 3) was used to screen a total imaginal disc cDNAlibrary prepared by A. Cowman. Screening approximately 2 million phageyielded three groups of cDNAs (five lats cDNAs; fifteen T1 cDNAs;fourteen T2 cDNAs). The sizes of the inserts in the lats cDNAs are asfollows: 5.6 kb in A2; 5.1 kb in B1; 1.1 kb in 9 and 4; and 0.9 kb inB3.

Genomic DNA from lats^(x1)/TM6B, lats^(a1-15)/TM6B, lats^(P1)/TM6B,lats^(e7-2)/TM6B, lats^(e78)/TM6B, lats^(e100)/TM6B, lats^(e119)/TM6Band lats^(e148)/TM6B flies was digested with a combination of the EcoRI,BamHI, BglII and XhoI restriction enzymes for Southern analysis.

DNA Sequencing

DNA sequence was determined by the dideoxy chain termination method(Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467) using Tagpolymerase (Perkin Elmer) and Sequenase (U.S. Biochemical Corp.). Thesequences of lats cDNAs were determined from both strands usingtemplates generated from plasmids containing EcoRI fragments insertedinto the pBlueScriptII vector. Templates generated from DNase 1 deletionsubclones were also used. The complete sequences of cDNAs A2 and 9 weredetermined; partial sequences were determined for cDNAs B1 and 4.Templates of genomic DNA were generated from plasmids containing EcoRIfragments and were sequenced on one strand using syntheticoligonucleotide primers. Mutant DNA from the lats^(a1) allele wasamplified with PCR reactions using synthetic oligonucleotide primers andcloned in the pBlueScript II vector for sequencing.

6.2. RESULTS

Screening for Overproliferation Mutations in Mosaic Animals

We have screened individuals carrying clones of cells that werehomozygous for either X-ray or P-element induced mutations foroverproliferation phenotypes. (FIG. 1B; Materials and Methods). Twotypes of overproliferation phenotypes were sought: a) Clones of mutantcells formed overproliferated, outgrowth tissues in anon-position-dependent fashion; b) Clones of mutant cells formed normalstructures, but proliferated faster than wild-type cells such that thesizes of the mutant clones were larger than their wt twin-spot clones.Three independent mutations were identified that caused the first typeof phenotype (FIG. 2A-2E). A mutation which was allelic to one of theoriginal mutations was later found to cause the second type of phenotype(see below). All three mutations in the first class caused embryonicand/or early larval lethality and they represented single alleles ofdifferent loci since they had different chromosome locations. One ofthem was identified among 215 randomly chosen lethal mutations in whicheach were caused by a P-element insertion in a different essential gene(Karpen and Spradling, 1992, Genetics 132:737-753; Berkeley DrosophilaGenome Center, personal communication). In addition to theseoverproliferation mutations, one P-induced mutation was found to causeboth unpatterned outgrowth and duplications of patterned structures inmosaic animals, suggesting that this mutation may not directly affectcell proliferation.

The lats Locus Is Defined by a Single Complementation Group of MutationsThat Cause Defects Throughout Development

The mutations caused different levels of overproliferation. One mutation(lats^(x1)) produced much more dramatic overproliferated clones than theones produced by the other mutations (FIG. 2A, 2B). The lats mutantclones induced in first instar larvae can be as large as ⅕ of the bodysize. Tumorous outgrowth caused by lats^(x1) was found in all thetissues that had been examined including eyes, legs, wings, heads,notums, antenna, and abdominal cuticles. The lats^(x1) mutation wasgenetically mapped in the 100A1-5 region and the locus was furtherdefined by a single complementation group of over fifty allelesincluding mutations induced by X-ray, EMS, P-element insertion andimprecise excision of the P-element (Table 2; Materials and Methods).

TABLE 2 The alleles of the lats locus* Phenotypes Phenotypes of ofmutant Representative No. of Alleles homozygous animals clones allelesalleles Strong Late embryonic and Large lats^(x1), lats^(a1), 14 early1st instar outgrowth lats^(a4) larval lethal Medium Late larval andpupal Large lats^(p1), lats^(e124) 16 lethal, normal size of outgrowthanimals Pupal lethal, giant Large lats^(e26-1) 3 animals outgrowth WeakSemi-viable and Mutant lats^(a10), lats^(e53-2) 17 viable: rough eyeclones outgrowth on head, larger or wing held-out, sterile normal insize *The various alleles of the lats gene are classified into threemain groups as indicated in the left column. Their phenotypes, displayedin either homozygous mutant animals or clones of mutant cells in mosaicanimals, are listed in the next two columns respectively: For a givenviable or semi-viable allele, the homozygous mutant animals display one,two, three, or all four of the listed phenotypes. Representative allelesand the numbers of alleles for each group are given in the two # rightcolumns. The origins of these alleles are described in the Material andMethods.

Removing the P-element insertion reverted the lethal chromosome intowild type, indicating the P-element insertion is responsible for themutant phenotype. Furthermore, five of the imprecise excision linescaused late embryonic and early larval lethality which were strongerthan the pupal lethality phenotype caused by the lats^(P1) mutation.These five excision lines failed to complement lats^(x1), butcomplemented the mutations in two other complementation groups(1(3)100Ab and zfh-1) in the 100A1-5 region, indicating that these twogenes were not affected by the excision alleles.

The lats alleles can be classified into three main groups (Table 2).Strong alleles caused homozygous animals to die at a late embryonicstage or shortly after hatching with no obvious cuticular defect.Mutations in the group of medium alleles cause lethality at differenttimes in larval and pupal development. This group was further dividedinto two subgroups because three of the excision alleles not only causedpupal lethality, but the sizes of the homozygous mutant animals werealso significantly larger than wt animals (FIG. 2C). The weak mutationscaused either one or a combination of the following phenotypes: held outwings with broadened blades, rough eye with ventral outgrowth, outgrowthon the dorsal-anterior region of the head and partial to completesterility (Table 2).

Proliferation defects were observed in both mutant clones in mosaicanimals and homozygous mutants. Clones of cells on the head that werehomozygous for strong or medium alleles formed unpatterned,overproliferated tissues with many lobes or folds. The mutant cellsseemed to be “budding out” of the surface to form new proliferationcenters or lobes (FIG. 2A, 2F, 2H). The sizes and the shapes of thesemutant cells were very irregular. Cells several times larger than theirneighbors were often seen in mutant clones, indicating problematic celldivision (FIG. 2F, 2G). Furthermore, lats mutant clones behaveddifferently from clones mutant for the previously identified Drosophilatumor suppressor genes such as dig, lgl and hyd. The dig, lgl or hydmutant cells proliferated slower than wt cells and thus, the mutantclones induced in first instar larvae were competed away during growthand did not form detectable clones in the adults (Bryant, 1987,Experimental and genetic analysis of growth and cell proliferation inDrosophila imaginal discs, in “Genetic Regulation of Development,” A. R.Liss, New York, pp. 339-372; Woods and Bryant, 1989; Dev. Biol.134:222-235; Mansfield et al., 1994, Dev. Biol. 165:507-526; AllenShearn, personal communication). In contrast, the lats mutant clonesinduced at similar developmental stages formed dramatic overproliferatedtissues, suggesting the mutant cells proliferated faster than wt cells.Consistent with this notion, clones of cells mutant for a weak latsallele (lats^(a10)) produced normal looking tissues, but the mutantclones were significantly larger than their wt twin-spot clones. Inhomozygous animals, the imaginal discs and the central nervous system inmany of the pupal lethal mutants were dramatically overproliferated(FIG. 2D, 2E). The discs lost the single layer of epithelial structureand formed multi-layer, deformed tissues. The lats overproliferationphenotype was not caused by prevention of differentiation. Cells in theoverproliferated mutant clones on the body differentiated and producedbristles and hairs, although the morphologies of these structures werenot wild type (FIG. 2I-2L). Careful examination of multiple mutantclones confirmed that lats caused mutant cells (w⁻ cells in the eye,y⁻bristles and enlarged-base hairs on the body) to overproliferate anddid not affect the surrounding wt tissues. Finally, the frequency ofoverproliferated clones was similar to wt clonal frequency induced withthe same FRT element, indicating that loss of the lats function alone issufficient to initiate the overproliferation process.

Cloning of the lats Gene

Genomic DNA from the 100A1-5 region was isolated using probes mapped tothis region (Materials and Methods). A restriction map of the relevantgenomic region is illustrated in FIG. 3. Genomic DNA flanking theP-insertion site (+7.5 to −4.2) was used to screen a total imaginal disccDNA library. A group of cDNAs corresponding to a 5.7 kb transcript(lats) was found to contain sequence from the region where the P-elementwas inserted (FIG. 3). Two other groups of cDNAs were also isolated (T1and T2). The 5.7 kb transcript was located in an intron of the T1 gene(FIG. 3). The intron-exon structure of the 5.7 transcription unit wasdetermined by Southern and sequence analysis of the cDNA clones and thecorresponding genomic DNA (Materials and Methods). The zfh-1 gene wasfound to be located at the left side of the 5.7 kb transcription unit(FIG. 3; Fortini et al., 1991, Mechanisms of Development 34:113-122).

In addition to lats^(P1), genomic DNA from the five strong excisionalleles was analyzed. All of them deleted exon sequences from the 5.7 kbtranscript and, in addition, three of them also deleted sequences in thenext transcript (T2; FIG. 3). Furthermore, DNA from the X-ray and EMSinduced mutants was analyzed with cDNA probes made from the 5.7 kb, T2and T1 transcripts. In two cases alterations were detected in the 5.7 kbtranscription unit: a 0.4 kb and a 0.3 kb deletions associated withlats^(a1) and lats^(a4), respectively (FIG. 3). The 446 bp deletion inlats^(a1) was revealed by sequencing. It removed codons 92 to 238 of theopen reading frame and caused a frame shift from codon 239 (FIG. 5).Finally, transformants containing a cDNA corresponding to the 5.7transcript driving by the hsp70 promoter rescued the lethality of bothstrong and medium lats alleles. These findings indicate that the 5.7 kbtranscription unit which correspond to the lats gene and strong latsalleles including lats^(a1) were either amorphic or nearly amorphicalleles.

The lats Gene Encodes a Putative Protein-Serine/Threonine Kinase

The 5.7 kb lats transcript was detected throughout development (FIG. 4)and in both adult males and females (data not shown). In addition,probes from the 5.7 kb transcript also detected a second transcript,which is about 1 kb shorter (4.7 kb), in young embryos (0-4hrs; FIG. 4)and in adult males and females. Northern analysis showed there was morematernally deposited 4.7 kb transcripts than 5.7 kb transcripts in youngembryos (0-2 hrs; FIG. 4). The 5.7 kb transcript became the dominantmessage at the embryonic stage (4-6 hrs), known to have zygotic geneexpression (FIG. 4). No effort was made to isolate cDNA clonescorresponding to the 4.7 kb transcript; thus the exact sequence of thisshort transcript is not known. However, a polyadenylation signalconsensus sequence was found at nucleotide position 4655-4660 in the 5.7kb transcript and in the corresponding genomic DNA (FIG. 5) and a 0.51kb probe from the 3′ end of the 5.7 kb transcript did not hybridize tothe 4.7 kb transcript while a 1 kb probe from the 5′ untranslated regionof the 5.7 kb transcript hybridized to both the 5.7 kb and 4.7 kbtranscripts. This suggests that the 4.7 kb transcript may be a truncatedversion of the 5.7 kb transcript. The genomic and cDNA sequencecorresponding to the 5.7 kb transcript was determined (Materials andMethods). The entire 5720 bp cDNA sequence, which is interrupted byseven introns, and the putative lats product (lats), deduced from thelong open reading frame, are illustrated in FIG. 5. An interestingfeature of the 5.7 kb transcript is the existence of a 141 bp segmentlocated in the 3′ untranslated region (FIG. 5), which is identical tothe first 141 bp of the 5′ untranslated region of the class I transcriptfrom the Drosophila phospholipase C gene, plc-21 (Shortridge et al.,1991, J. Biol. Chem. 266:12474-12480). The functional significance ofthis sequence motif is unknown. It could be a regulatory target sequencethat is shared by both genes.

There are 34 differences between the lats cDNA and genomic sequences and31 of them do not affect the deduced amino acid sequence. In theremaining three differences, one changes the serine 206 in cDNA into acysteine. The second change in the genomic sequence adds an additionalglutamine in the poly-glutamine opa repeat (FIG. 6; Wharton et al.,1985, Cell 40:55-62). The third is the addition of a fifteen bp sequencein the genomic DNA after the nucleotide 2644 of the cDNA. This sequencecould be translated into another copy of the Arg-Glu-Arg-Asp-Gln (partof SEQ ID NO:2) peptide. However, this sequence is not present in thetwo independent cDNA clones that were sequenced.

The predicted lats product contains 1099 amino acid residues. The kinasedomain of lats is more similar to protein-serine/threonine kinases thanto protein-tyrosine kinases, especially in the sequences of the domainsVI and VIII defined by Hanks et al. (1988, Science 241:42-52);protein-serine/threonine kinase consensus in domain VI:Asp-Leu-Lys-Pro-Glu-Asn (SEQ ID NO:9). Lats sequence in domain VI:Arg-Asp-Ile-Lys-Pro-Asp-Asn (836-842) (part of SEQ ID NO:2);protein-serine/threonine kinase consensus in domain VIII:Gly-Thr/Ser-X-X-Tyr/Phe-X-Ala-Pro-Glu (SEQ ID NO:10). Lats sequence indomain VIII: Gly-Thr-Pro-Asn-Tyr-Ile-Ala-Pro-Glu (917-925) (part of SEQID NO:2). The C-terminal half of lats shares extensive sequencesimilarity with a group of six proteins including the Dbf20 and Dbf2cell cycle protein-ser/thr kinases from Saccharomyces cerevisiae(Johnston et al., 1990, Mol. Cell. Biol. 10:1358-1366; Toyn et al.,1991, Gene 104:63-70; Toyn and Johnston, 1994, EMBO J. 13:1103-1113),and the COT-1 putative protein kinase from Neurospora crassa (Yarden etal., 1992; EMBO J. 11:2159-2166) (FIG. 6A, 6B). The sequence similaritybetween the kinase domains of lats and these proteins (39-49% identity)is much higher than the sequence similarity observed between thedifferent subgroups of protein-ser/thr kinases (20-25% identity; Hankset al., 1988, Science 241:42-52). However, there is an insertion ofabout 40 amino acid residues within the kinase domains of theseproteins, sharing little sequence similarity (denoted by a black bar inFIG. 6B). The human myotonic dystrophy protein kinases (MDPK) also havesignificant similarity with the C-terminal region of lats (Brook et al.,1992, Cell 68:799-808; Fu et al., 1993; Science 260:235-238, Mahadevanet al., 1993, Hum. Mol. Genet. 2:299-304), but their kinase domains donot contain this ˜40 amino acid insertion. In addition, lats and theseproteins also share significant levels of sequence similarity in the tworegions (each contains ˜100-150 amino acids) flanking the kinase domain(20-28% identity; FIG. 6A, 6B). In the case of Dbf20, its entiresequence except for the 20 C-terminal most residues can be aligned withlats, indicating lats is a close relative of Dbf20. A poly-glutamine oparepeat is located near the middle of the protein (FIG. 5; Wharton etal., 1985, Cell 40:55-62). The N-terminal half of lats contains manyshort homopolymeric runs including poly-proline which makes up about 15%of the residues. At least one of the proline-rich stretches closelymatches the consensus of SH3-binding sites (FIG. 3B; Ren et al., 1993,Science 259:1157-1161), raising the possibility that it may interactwith SH3-containing proteins. No putative signal sequence appears in thelats protein, indicating that it is an intracellular protein.

6.3. DISCUSSION

Screening for Mutations in Mosaic Animals to Identify and StudyPotential Tumor Suppressors

The comparison between mosaic flies and tumor patients is simplistic yetuseful. Tumor patients contain wt tumor suppressor genes in most oftheir cells and only small groups of cells sustain mutations in tumorsuppressors. We have searched for recessive overproliferation mutationsin mosaic animals. Flies that carry somatic cells mutated for tumorsuppressors or negative regulators of cell proliferation are viable, yetthe overproliferation mutant phenotype is readily identifiable.Therefore, mosaic flies, which are in a fashion analogous to tumorpatients, provide a mean to screen for potential tumor suppressors.Three overproliferation mutations were identified in our screen. Theywere not identified as “interesting” mutations in screens for embryoniclethal mutations. Identifying overproliferation mutations in homozygousmutant larvae and pupae is not only biased against embryonic lethals,but also laborious, since it requires establishment of individual linesbefore examining the potential phenotypes. Further screens foroverproliferation mutations in mosaic animals will allow us to identifyother important players in pathways that negatively regulate cellproliferation.

The overproliferation phenotypes that we observed were caused by loss offunction in a single gene. In humans, it was suggested that mostretinoblastomas are caused by defects in a single tumor suppressor(Knudson, 1971, Proc. Natl. Acad. Sci. USA 68:820-823). On the otherhand, evidence indicates that tumorigenesis in other human tissues(e.g., colon cancer) is a multistep process which involves inactivationof more than one gene (Fearon and Vogelstein, 1990, Cell 61:759-767;Vogelstein and Kinzler, 1993, Trends Genet. 9:138-141).Overproliferation caused by defects in multiple genes is unlikely to bedetected in our screens unless these genes are located on the samechromosome arm. To identify this type of gene, one could perform amodified mosaic screen which induces clones of cells to becomehomozygous for more than one mutagenized chromosome arm.

lats Affects Many Tissues Throughout Development

The lats gene is genetically defined by a single complementation groupthat consists of various alleles causing a wide range of defects.Different alleles were found to cause lethality at almost every stageduring development: embryo, early larvae, late larvae, early pupae, latepupae and pharate-adult. The embryonic lethality occurs in the pharatefirst instar stage. The early embryonic requirements for lats could wellbe masked by the wt products that are maternally deposited in the egg.Weak lats alleles produce viable animals with phenotypes ranging fromrough eye to sterility. The lats transcripts were detected throughoutdevelopment up to adult stage, consistent with the observation that latsmutants affect all these stages. Although mutations at lats cause manydefects, affecting cell proliferation could cause most of the phenotypesincluding overproliferation in mutant clones, lethality at the variousstages, tissue overproliferation on the head, broadened wing blade, andsterility in homozygous mutants. However, phenotypes such as extracuticle deposits and malformed bristles and hairs are evidence ofdefects in differentiation.

The different behavior of the lats mutant clones and clones mutant forother previously identified Drosophila tumor suppressors is interesting.Cells mutant for dig, lgl or hyd seem to fail to receive growthregulation signals. They proliferated slower than wt cells during larvalstages when the cells were instructed to proliferate, and they failed toterminate proliferation in late larval and pupal stages when the wtcells have ceased proliferation. On the other hand, the lats mutantclones induced during the larval stages were overproliferated, and laterthe mutant cells on the body were differentiated to form adult cuticularstructures. Thus, lats could be a negative regulator that monitors therate of proliferation.

The lats gene is located in a complex region. The 5′ end of the lats 5.7kb transcript (cDNA) is only about 550 bp away from the T2 transcriptand its 3′ end is about 1.5 kb away from the zfh-1 transcript.Furthermore, all three of these closely located transcripts are locatedin an intron of the T1 transcription unit. Thus, a sizable deletion inthe 5.7 kb transcription unit could affect the function of any of thegenes in the region, which makes it difficult to determine whichtranscript is responsible for the lats phenotype. The fact thatP-element transform lines carrying a cDNA from the 5.7 kb transcriptunder the hsp70 promoter rescued all types of lats alleles demonstratedthat the 5.7 kb transcription unit is the lats gene.

The lats Putative Protein-Ser/Thr Kinase Shares Homology With ProteinsThat Are Involved in Regulation of Cell Cycle and Growth in BuddingYeast and Neurospora

All 11 subdomains of the kinase domain that are found in previouslyidentified protein kinases (Hanks et al., 1988, Science 241:42-52) areconserved in lats. This predicts that lats is a protein kinase.Furthermore, the sequence comparisons suggest lats to be a ser/thrkinase as the lats kinase domain is more similar to protein-ser/thrkinases than to protein-tyr kinases. The C-terminal half of lats sharesextensive sequence similarity with a group of six proteins. Mutationsare known for three of these genes and in each case they affect eithercell cycle or growth. The cot-1 (colonial temperature sensitive-1) geneof Neurospora was identified by a temperature sensitive mutant thatcauses compact colony growth (Mitchell and Mitchell, 1954, Proc. Natl.Acad. Sci. USA 40:436-440; Galsworthy, 1966, Diss. Abstr. 26:6348).Wild-type filamentous ascomycete Neurospora grows on solid media bycontinuous hyphal elongation and branching to form spreading colonies.Strains lacking functional cot-1 gene are viable, but their hyphaebranch extensively, resulting in compact colonial growth (Yarden et al.,1992, EMBO J. 11:2159-2166). This extensive branching phenotype issomewhat similar to the growth property of the lats mutant clones: thelats mutant cells continue to “bud” out of the surface to form newproliferation lobes. Another homologous gene, the DBF2 gene of thebudding yeast, was identified in a genetic screen for mutations causingdefects in DNA synthesis (Johnston and Thomas, 1982, Mol. Gen. Genet.186:439-444). The temperature sensitive alleles of DBF2 were found toboth delay the initiation of S phase and also to arrest the cell cycleduring nuclear division (Johnston et al., 1990, Mol. Cell. Biol.10:1358-1366). The DBF20 gene was identified through cross hybridizationwith DBF2 DNA (Toyn et al., 1991, Gene 104:63-70). Strains carryingdeletions for either DBF2 or DBF20 are viable; however, deleting bothgenes in the same strain causes lethality. The kinase activities of bothproteins have been shown to be specific for serine/threonine residuesand are regulated during the cell cycle (Toyn and Johnston, 1994, EMBOJ. 13:1103-1113). In the case of Dbf20, its entire sequence except the20 most C-terminal residues can be aligned with lats. The mutantphenotype of lats and its sequence homology with the cell cycle proteinkinases is consistent with the notion that lats might be directlyinvolved in regulation of the cell cycle. The N-terminal half of latscontains many proline-rich stretches and at least one of them closelymatches the consensus sequence of SH3 binding sites (Ren et al., 1993,Science 259:1157-1161), raising the possibility that this region couldbe a regulatory domain for the lats kinase, which binds to SH3domain-containing proteins.

In recent years, many protein kinases have been identified to beinvolved in regulation of the cell cycle and cell proliferation. WhileWeel is an inhibitor of the Cdc2 kinase (Russell and Nurse, 1987, Cell49:559-567; Featherstone and Russell, 1991, Nature 349:808-811), allother previously identified protein kinases are positive regulators ofcell proliferation. They are either required for completion of the cellcycle or for signalling cells to proliferate. Lats is the firstpredicted protein-ser/thr kinase that has been shown to causeoverproliferation when its function is removed. Studies of lats andother overproliferation mutations in Drosophila will provide a betterunderstanding of how cell proliferation is regulated during developmentand how mutations could lead to abnormal growth.

7. ISOLATION AND CHARACTERIZATION OF MAMMALIAN LATS HOMOLOGS

As described herein, we have cloned and sequenced both mouse and humanlats homologs.

7.1. ISOLATION AND CHARACTERIZATION OF MOUSE LATS HOMOLOGS

cDNA clones for two different lats homologs in mice were obtained asfollows.

Screening of Mouse Homologs: Probe: A 2.2 kb BamHI fragment containingthe kinase domain of the Drosophila lats gene was labeled with ³²P byrandom labeling Library: Newborn mouse brain lambda ZAP cDNA libraryfrom Stratagene Hybridization Condition: 45 C., overnight in 6x SSC 5xDenhart's 0.5% SDS (sodium dodecyl sulfate) 100 μg/ml salmon sperm DNAWash: 50° C., 30 min. × 4, in 2x SSC 0.1% SDS Results: Three positiveclones were identified. (M41 clone for the m-lats gene, and M51 and M31clones for the m-lats 2 gene.)

Two different mouse lats homologs, termed m-lats and m-lats2,respectively, were isolated and sequenced. Both the m-lats and m-lats2clones are missing a small amount of the 5′ end of their respectivegenes. The cDNA sequence (SEQ ID NO:5) and deduced protein sequence (SEQID NO:6) of m-lats are shown in FIG. 7. The cDNA sequence (SEQ ID NO:7)and deduced protein sequence (SEQ ID NO:8) of m-lats2 are shown in FIG.8.

Portions of both the m-lats and m-lats2 cDNAs were used as probes toscreen a mouse genomic library, under standard hybridization conditions.Genomic clones for both m-lats and m-lats2 have been isolated thatcontain most of the coding regions of these genes.

7.2. ISOLATION AND CHARACTERIZATION OF HUMAN LATS HOMOLOGS

cDNA clones for at least one human lats homolog were obtained asfollows.

Screening of Human Homologs (moderately stringent conditions): Probe: A2.1 kb PstI fragment containing the kinase domain of the m-lats gene waslabeled with ³²P by random labeling Library: Fetal human brain lambdagt10 cDNA library from Contech Hybridization Condition: 55 C., overnightin 6x SSC 5x Denhart's 0.5% SDS 100 μg/ml salmon sperm DNA Wash: 60° C.,30 min. × 4, in 1x SSC 0.1% SDS Results: About 20 positive clones wereidentified for the h-lats 2 gene.)

One human lats homolog, termed h-lats, was isolated and sequenced. ThecDNA sequence (SEQ ID NO:3) and deduced protein sequence (SEQ ID NO:4)of h-lats are shown in FIG. 9. The deduced protein sequence isfull-length. The complete coding sequence of the h-lats cDNA wasinserted into a bacterial cloning vector (derived from Bluescript(KS)-vector; Stratagene) to form plasmid pBS(KS)-h-lats (FIG. 10). Thetotal size of pBS(KS)-h-lats is 6.96 kb.

A h-lats cDNA fragment was used as a probe under conditions of moderatestringency to screen a human genomic cosmid library. Genomic h-latsclones were isolated. Over 70 kb of the genomic h-lats sequence has beenisolated; the isolated sequences include all of the h-lats codingsequence (but not all the exon sequences).

An m-lats2 cDNA fragment was used as a probe to screen a human genomicphage library under the conditions described above, except thathybridization was carried out at 50° C., and washing was carried out at55° C. with 2×SSC, 0.1% SDS. Two genomic h-lats clones have beenisolated that specifically hybridize to m-lats2 cDNA probes and do nothybridize to m-lats and h-lats cDNA probes.

8. CONSERVATION OF SEQUENCES AND DOMAIN STRUCTURE AMONG LATS HOMOLOGS OFDIFFERENT SPECIES

Comparison of the sequences of Drosophila lats, h-lats, m-lats, andm-lats2 showed a startlingly high degree of sequence conservation, bothoverall and within domains of the lats protein. An alignment of theh-lats (SEQ ID NO:4) and m-lats (SEQ ID NO:6) protein sequences is shownin FIG. 11. The overall amino acid sequence identity between h-lats andm-lats is 93%. An alignment of the h-lats (SEQ ID NO:4) and m-lats2 (SEQID NO:8) protein sequences is shown in FIG. 12.

Homologous domains (i.e., domains conserved) between the different latshomologs were identified. FIG. 13 presents an alignment of the h-latsprotein sequence (SEQ ID NO:4) and the Drosophila lats protein sequence(SEQ ID NO:2), and indicates the domains identified as conserved amongthe lats homologs from the various species.

The identified domains were as follows:

(1) Lats C-terminal domain 3 (LCD3)

The last three amino acids (VYV) are completely conserved in all fourhomologs including Drosophila lats, h-lats, m-lats, and m-lats2.

(2) Lats C-terminal domain 2 (LCD2)

amino acid residues h-lats 1077-1086 Drosophila lats 1075-1084

This domain is completely conserved in all four homologs includingDrosophila lats, h-lats, m-lats, and m-lats2 (10/10 identical residues).

(3) Lats C-terminal domain 1 (LCD1)

amino acid residues h-lats 1032-1043 Drosophila lats 1035-1047

This domain is completely conserved among Drosophila lats, h-lats, andm-lats (12/12 identical), and is highly conserved between any of theforegoing and m-lats2 (11/12 identical).

(4) Kinase domain

amino acid residues h-lats 703-1014 Drosophila lats 711-1018

This domain is highly conserved among the four homologs (76% identicalbetween Drosophila lats and h-lats; 99% identical between h-lats andm-lats; 83% identical between h-lats and m-lats2).

A potential phosphorylation residue in Drosophila lats and the mammalianhomologs that could lead to the activation of the lats kinases afterphosphorylation was identified.

Activities of protein kinases are often regulated by varying thephosphorylation state of specific serine, threonine, and tyrosineresidues. Phosphorylation of a serine or threonine within twentyresidues upstream of an Ala-Pro-Glu consensus in subdomain eight of thekinase domain, is often required for catalytic activities of manyprotein-ser/thr kinases (Hanks et al., 1988, Science 241:42-52). Forexample, Thr167 and Thr197 are phosphorylated in Cdc2 of fission yeastand in the cardiac muscle adenosine 3′,5′-phosphate dependent proteinkinase, respectively (Ducommun et al., 1991, EMBO J. 10:3311-3319; Gouldet al., 1991, EMBO J. 10:3297-3309; Shoji et al., 1983, Biochem.22:3702-3709). A ser residue in a similar position of the lats kinasedomain is conserved in Drosophila lats, h-lats, m-lats, and m-lats2(Ser914 in Drosophila lats; Ser909 in h-lats). Thus, the activities ofDrosophila lats and its mammalian homologs may be regulated byphosphorylation of this ser residue.

(5) Lats flanking domain (LFD)

amino acid residues h-lats 607-702 Drosophila lats 612-710

LFD is a domain that flanks and is amino-terminal to the kinase domain.This domain is highly conserved between Drosophila lats and h-lats (68%identical) and is also highly conserved between h-lats and m-lats2 (71%identical). This domain is completely conserved between h-lats andm-lats (100% identical).

(6) Lats split domain 1 (LSD1)

amino acid residues LSD1 Drosophila-lats 365-392 LSD1 anterior (LSD1a)h-lats 328-334 LSD1 posterior (LSD1p) h-lats 498-518

Certain lats domains have been termed split domains because the amino-(anterior) and carboxy- (posterior) portions of the domain appearseparated from each other in at least one of the lats homologs. Splitdomains may constitute discontinuous binding/functional regions (e.g.,brought together by tertiary structure). The LSD1a subdomain iscompletely conserved among Drosophila lats, h-lats, and m-lats (7/7identical), and is not conserved in m-lats. The LSD1p subdomain isconserved between the four homologs (14/21 identical among Drosophilalats, h-lats, and m-lats; 13/21 identical between h-lats and m-lats2).The LSD1a and LSD1p subdomains are adjacent to each other in Drosophilalats and are separated in the mammalian homologs.

(7) Lats split domain 2 (LSD2)

amino acid residues LSD2 Drosophila-lats 536-544 LSD2 anterior (LSD2a)h-lats 28-31 LSD2 posterior (LSD2p) h-lats 555-559

Both the LSD2a and LSD2p subdomains are completely conserved among thefour homologs. However, the two subdomains are adjacent to each other inDrosophila lats and are separated in the mammalian homologs.

(8) Putative SH3-binding domain (SH3-binding)

amino acid residues h-lats 247-268 Drosophila lats 196-217

This domain is highly conserved among Drosophila lats, h-lats, andm-lats (10/22 identical), and does not exist in m-lats2.

The opa domain does not appear in the mammalian lats homologs.

9. FUNCTIONAL INTERCHANGEABILITY OF THE HUMAN AND DROSOPHILA LATSHOMOLOGS

9.1. OVEREXPRESSION OF HUMAN LATS OR OF DROSOPHILA LATS CAUSES ASMALLER, ROUGH EYE IN DROSOPHILA

Overexpression of lats and h-lats in the developing Drosophila eye wascarried out. The Drosophila lats cDNA and h-lats cDNA were each clonedinto the pGMR P-element vector. This vector was constructed by Bruce Hayand Gerald M. Rubin at the University of California at Berkeley, andwill direct the expression of a cDNA of interest in the posterior regionof the developing third instar larval eye imaging disc of Drosophila.Ten independent transformant lines for each of the pGMR-lats andpGMR-h-lats constructs were generated. The adult eyes of all these linesdisplayed a small-rough eye phenotype (eyes smaller than normal, withirregular, rough appearance). This indicates that both lats and h-latsgenes have the same biological effect when they are overexpressed in thedeveloping Drosophila eye.

9.2. HUMAN H-LATS GENE CAN REPLACE THE DROSOPHILA HOMOLOGS TO PREVENTDEATH IN DROSOPHILA ANIMALS HAVING MUTANT DROSOPHILA LATS

The Drosophila lats cDNA was cloned into the pCaSpeR-hs vector (Thummeland Pirrotta, 1992, Drosophila Inform. Service 71:150) for germ linetransformation of Drosophila. Three of the transformed lines were testedand were able to rescue the lethality of the lats^(a1)/lats^(x1),lats^(P1) and lats^(e26-1) animals after one hour heat shock for every24 hours during larval and pupal development. The human h-lats CDNA (ina XhoI (blunted)-XbaI fragment) from pBS(SK)-h-lats (FIG. 10) was clonedinto the HpaI-XbaI sites of the pCaSpeR-hs vector, to produce plasmidpCaSpeR-hs-h-lats (FIG. 14). Plasmid pCaSpeR-hs-h-lats was used for germline transformant. Three of the pCaSpeR-hs-h-lats transformant lineswere tested and were able to rescue the lethality of the lats^(P1) andlats^(e26-1) animals under the same conditions used in rescuingexperiments for the Drosophila gene.

10. HUMAN LATS EXPRESSION IS FOUND IN ALL NORMAL TISSUES TESTED AND ISABSENT IN A LARGE NUMBER OF TUMOR CELL LINES

10.1. HUMAN LATS EXPRESSION IN NORMAL TISSUES

The expression of human lats RNA was investigated in various adulttissues. A 1.2 kb BamHI fragment of the h-lats cDNA was used as a³²P-labeled probe for Northern analysis. Hybridization was to a nylonmembrane containing polyA⁺ RNA from various human fetal and adulttissues, obtained from Clontech. The Northern analysis was carried outaccording to the recommended instructions of the manufacturer(Clontech). The results are shown in FIG. 15. h-lats was expressed inevery tissue tested (fetal brain, fetal lung, fetal liver, fetal kidney,adult spleen, adult thymus, adult prostate, adult testis, adult ovary,adult small intestine, adult colon, and adult blood leukocytes).Expression was higher in fetal tissues than in adult tissues.

10.2. HUMAN LATS EXPRESSION IN VARIOUS TUMOR CELL LINES

The ³²P-labeled BamHI fragment of h-lats was used as a probe forNorthern analysis, for hybridization to total RNAs isolated from 42different human tumor cell lines (obtained from the American TypeCulture Collection, Rockville, MD). No h-lats expression was detected in20 of the tumor lines (48%). The name and tissue origin of the tumorcell lines tested, and the results of the Northern analysis arepresented in Table 3.

TABLE 3 Expression detected by Northern analyses Name of tumor linesTumor Origin YES NO 5637 Bladder × RT4 Bladder ±* HT-1376 Bladder ×HT-1197 Bladder × BT-20 Breast × BT-474 Breast × ZR-75-1 Breast ×ZR-75-30 Breast × BT-549 Breast × MDA-MB-453 Breast × MDA-MB-435S Breast× HBL-100 Breast × LoVo Colon × HT-29 Colon × HCT116 Colon × LS 180Colon × DLD-1 Colon × WiDr Colon × SW480 Colon × Caco-2 Colon ± HEL92.1.7 Erythroleukemia × MOLT-4 Leukemia × CEM-CM 3 Leukemia × K-562Leukemia × Jurkat Leukemia × HUT 78 Lymphoma × SK-LU-1 Lung × A-427 Lung× Calu-1 Lung × NCI-H69 Lung × SK-MEL-3 Melanoma × SK-MEL-28 Melanoma ×SK-MEL-31 Melanoma × MIA PaCa-2 Pancreas × BxPC-3 Pancreas × Hs 700TPancreas × Hs 766T Pancreas × RD Sarcoma × A-204 Sarcoma × AN3 CAUterine × SK-UT-1 Uterine × HEC-1-A Uterine ± *weak signal

Thus, 48% of the tumor cell lines tested had no detectable h-latsexpression, whereas 100% of the normal tissues tested had detectableh-lats expression. It should be noted that the 48% figure may be anunderestimate of the actual number of tumor cell lines that haddecreased lats protein level or activity relative to normal tissue,since while lack of lats RNA (i.e., a transcriptional block) allows theconclusion that no lats protein is made, tumor cells that expressed thelats RNA may still have had no or low lats protein levels and/oractivity due to the possible existence of a translational block or thepresence of mutation(s) in an expressed lats protein.

11. DEPOSIT OF MICROORGANISM

Bacteria strain E. coli TG2 containing plasmid pBS(KS)-h-lats wasdeposited on Mar. 24, 1995 with the American Type Culture Collection,1201 Parklawn Drive, Rockville, Md. 20852, under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedures, and assignedAccession No. 69769.

The present invention is not to be limited in scope by the microorganismdeposited or the specific embodiments described herein. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying figures. Such modifications are intended tofall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

16 5720 base pairs nucleic acid double unknown cDNA CDS 1103..4402 1ATCTAGCACG ACGGCAGCAA CAAAACCACG AATTAATTTT ACTAAATTTA AGCCAAACGC 60GCATCGGAAA TGCCTGAAAA TGCGATTGAA TGCACGCGAA AAGTGATGGG TTGCGAACGC 120GAGTGAATCA AGTGAAAATA CGTCGGCAAA TATCAGCGAA TTGTCGTCAA AAGGCAAGGA 180AAAACGGAGA AAAAGAGGAA AAGCAATAAG TGCCGTGTGT GGGAAACGCG AAAAAGGCGA 240GAACAAAGAG GCGAAAAGCG AGGAAATTGC GTGGAAAACG TGGAAAACGC GAAGAAGCGA 300AGCTCCAAGT TGGCCGCCAT CGATTCGTGC GTAGGATCAA TTAAGATTCC GAGTGGTCGA 360GAATCGGCTC AAATCAAATT AAAATCAACT AATATTTTGG TATTCAGATA TTCAAATGGA 420ATTCATTCAT CGCCTGCGAC TTTTATTCGG ATCTGCCAAC TATTTTTGAA TTTGAATTGT 480GTGTCTGCGG CTGGCGCAGA ATCTCTGATA AAGCAGAGGA ATAAAATCGG AAGAACAACA 540AATACAAATA CAAATGAAAT GCGGGGAGCA GTATTTACAT GCCAAATGAA TGCTGGATAG 600GCGAAAGGGG GGGTTTCTCT TATAATGCAA ATGTGAATGT GAATGCGAAT GCGAATGCGA 660GTGGAAGAAT TCCCGGCGCG AGTGATAAAT AATCCGACGA CAAACAAAGC AGAAGCCTAC 720ACCGCGAGAA AGAGCAGCGC AAACACAATT ATCTTTATTG AGAGCAACAA TATCAAGATC 780GAGATAATAA AGCATCCTAA AACCCGCGCC TTAGTTCGTT TTAGTCTCGC CACGGATATA 840GATATTCAAA GGCAAAAAGG TGGTGTCGGC ATCGCCAGAC AAACAAGTAA AGCATCTATT 900TCATACAAAA CAACCAATTA AATAATAATA AAAATAATAA TAATCGTAGA GAGGCAGAGC 960CAAATCAAAT TCCCGGCCGC CGATGTGCCC CAGTGTGTGT GCGTGTGTGT GTGTGTGTGC 1020TGTGCTGTGC TGTGCGAGTG TTAGTGTGCG GAGCATTTCT GTGATATGAG TGCTAAATGC 1080CACAGGGCGA AGCAGCAGCA TC ATG CAT CCA GCG GGC GAA AAA AGG GGC GGT 1132Met His Pro Ala Gly Glu Lys Arg Gly Gly 1 5 10 CGC CCC AAT GAT AAA TACACG GCG GAA GCC CTC GAG AGC ATC AAG CAG 1180 Arg Pro Asn Asp Lys Tyr ThrAla Glu Ala Leu Glu Ser Ile Lys Gln 15 20 25 GAC CTA ACC CGA TTT GAA GTACAA AAT AAC CAT AGG AAT AAT CAG AAT 1228 Asp Leu Thr Arg Phe Glu Val GlnAsn Asn His Arg Asn Asn Gln Asn 30 35 40 TAC ACA CCT CTG CGA TAC ACG GCGACC AAC GGA CGC AAC GAT GCA CTT 1276 Tyr Thr Pro Leu Arg Tyr Thr Ala ThrAsn Gly Arg Asn Asp Ala Leu 45 50 55 ACT CCT GAC TAT CAC CAC GCC AAG CAGCCG ATG GAG CCG CCA CCC TCC 1324 Thr Pro Asp Tyr His His Ala Lys Gln ProMet Glu Pro Pro Pro Ser 60 65 70 GCC TCT CCT GCT CCG GAC GTG GTC ATA CCGCCG CCG CCC GCC ATT GTA 1372 Ala Ser Pro Ala Pro Asp Val Val Ile Pro ProPro Pro Ala Ile Val 75 80 85 90 GGT CAG CCC GGA GCC GGC TCC ATA TCC GTATCC GGT GTG GGC GTT GGA 1420 Gly Gln Pro Gly Ala Gly Ser Ile Ser Val SerGly Val Gly Val Gly 95 100 105 GTG GTG GGT GTG GCG AAC GGA CGT GTG CCAAAG ATG ATG ACG GCC CTA 1468 Val Val Gly Val Ala Asn Gly Arg Val Pro LysMet Met Thr Ala Leu 110 115 120 ATG CCA AAC AAA CTG ATC CGG AAG CCG AGCATC GAA CGG GAC ACG GCG 1516 Met Pro Asn Lys Leu Ile Arg Lys Pro Ser IleGlu Arg Asp Thr Ala 125 130 135 AGC AGT CAC TAC CTG CGC TGC AGT CCG GCTCTG GAC TCC GGA GCC GGT 1564 Ser Ser His Tyr Leu Arg Cys Ser Pro Ala LeuAsp Ser Gly Ala Gly 140 145 150 AGC TCC CGA TCG GAC AGC CCC CAT TCG CACCAC ACC CAC CAG CCG AGC 1612 Ser Ser Arg Ser Asp Ser Pro His Ser His HisThr His Gln Pro Ser 155 160 165 170 TCG AGG ACG GTG GGT AAT CCA GGT GGAAAT GGT GGA TTT TCT CCG TCG 1660 Ser Arg Thr Val Gly Asn Pro Gly Gly AsnGly Gly Phe Ser Pro Ser 175 180 185 CCA AGC GGT TTC AGT GAG GTG GCT CCACCG GCG CCG CCG CCA CGC AAT 1708 Pro Ser Gly Phe Ser Glu Val Ala Pro ProAla Pro Pro Pro Arg Asn 190 195 200 CCC ACC GCC TCC AGC GCG GCC ACG CCCCCA CCG CCA GTG CCG CCC ACC 1756 Pro Thr Ala Ser Ser Ala Ala Thr Pro ProPro Pro Val Pro Pro Thr 205 210 215 AGC CAG GCG TAC GTG AAG CGG CGA TCACCG GCC CTG AAC AAC CGC CCG 1804 Ser Gln Ala Tyr Val Lys Arg Arg Ser ProAla Leu Asn Asn Arg Pro 220 225 230 CCG GCG ATA GCG CCA CCC ACT CAG CGAGGC AAC TCA CCT GTA ATA ACC 1852 Pro Ala Ile Ala Pro Pro Thr Gln Arg GlyAsn Ser Pro Val Ile Thr 235 240 245 250 CAA AAC GGG CTG AAG AAC CCG CAGCAG CAG TTG ACG CAG CAG CTG AAG 1900 Gln Asn Gly Leu Lys Asn Pro Gln GlnGln Leu Thr Gln Gln Leu Lys 255 260 265 TCC CTG AAC CTA TAC CCA GGC GGAGGC AGT GGA GCA GTG GTG GAG CCA 1948 Ser Leu Asn Leu Tyr Pro Gly Gly GlySer Gly Ala Val Val Glu Pro 270 275 280 CCG CCG CCC TAC CTA ATT CAA GGCGGA GCC GGA GGA GCA GCA CCG CCG 1996 Pro Pro Pro Tyr Leu Ile Gln Gly GlyAla Gly Gly Ala Ala Pro Pro 285 290 295 CCG CCA CCA CCC AGT TAC ACG GCCTCC ATG CAG TCG CGG CAG TCG CCC 2044 Pro Pro Pro Pro Ser Tyr Thr Ala SerMet Gln Ser Arg Gln Ser Pro 300 305 310 ACA CAA TCC CAA CAA TCG GAC TACAGG AAA TCC CCG AGC AGT GGG ATA 2092 Thr Gln Ser Gln Gln Ser Asp Tyr ArgLys Ser Pro Ser Ser Gly Ile 315 320 325 330 TAC TCG GCC ACC TCG GCG GGCTCG CCG AGC CCC ATA ACT GTG TCG CTG 2140 Tyr Ser Ala Thr Ser Ala Gly SerPro Ser Pro Ile Thr Val Ser Leu 335 340 345 CCG CCG GCG CCG CTG GCG AAGCCA CAA CCA CGA GTC TAC CAG GCC AGG 2188 Pro Pro Ala Pro Leu Ala Lys ProGln Pro Arg Val Tyr Gln Ala Arg 350 355 360 AGT CAG CAG CCG ATC ATC ATGCAG AGT GTG AAG AGC ACG CAG GTC CAA 2236 Ser Gln Gln Pro Ile Ile Met GlnSer Val Lys Ser Thr Gln Val Gln 365 370 375 AAG CCC GTG CTG CAA ACA GCAGTG GCG CGC CAA TCG CCA TCG AGT GCC 2284 Lys Pro Val Leu Gln Thr Ala ValAla Arg Gln Ser Pro Ser Ser Ala 380 385 390 TCG GCC AGC AAT TCA CCA GTCCAC GTG CTG GCC GCT CCA CCC TCT TAC 2332 Ser Ala Ser Asn Ser Pro Val HisVal Leu Ala Ala Pro Pro Ser Tyr 395 400 405 410 CCT CAG AAG TCC GCG GCAGTG GTG CAG CAG CAG CAA CAG GCA GCA GCG 2380 Pro Gln Lys Ser Ala Ala ValVal Gln Gln Gln Gln Gln Ala Ala Ala 415 420 425 GCG GCC CAC CAG CAG CAGCAT CAG CAC CAG CAA TCC AAA CCA CCA ACG 2428 Ala Ala His Gln Gln Gln HisGln His Gln Gln Ser Lys Pro Pro Thr 430 435 440 CCA ACC ACA CCG CCC TTGGTG GGT CTG AAC AGC AAG CCC AAT TGC CTG 2476 Pro Thr Thr Pro Pro Leu ValGly Leu Asn Ser Lys Pro Asn Cys Leu 445 450 455 GAG CCA CCG TCC TAT GCCAAG AGC ATG CAG GCC AAG GCG GCC ACG GTG 2524 Glu Pro Pro Ser Tyr Ala LysSer Met Gln Ala Lys Ala Ala Thr Val 460 465 470 GTA CAG CAG CAG CAA CAGCAG CAG CAA CAA CAG CAG GTC CAG CAG CAG 2572 Val Gln Gln Gln Gln Gln GlnGln Gln Gln Gln Gln Val Gln Gln Gln 475 480 485 490 CAG GTG CAA CAG CAGCAG CAA CAG CAG CAA CAG CAA CTG CAG GCC TTG 2620 Gln Val Gln Gln Gln GlnGln Gln Gln Gln Gln Gln Leu Gln Ala Leu 495 500 505 AGG GTG CTC CAG GCACAG GCT CAG AGG GAG CGG GAT CAA CGG GAG CGG 2668 Arg Val Leu Gln Ala GlnAla Gln Arg Glu Arg Asp Gln Arg Glu Arg 510 515 520 GAA CGG GAT CAG CAGAAG CTG GCC AAC GGA AAT CCT GGC CGG CAG ATG 2716 Glu Arg Asp Gln Gln LysLeu Ala Asn Gly Asn Pro Gly Arg Gln Met 525 530 535 CTT CCG CCG CCG CCCTAT CAG AGC AAC AAC AAC AAC AAC AGC GAG ATC 2764 Leu Pro Pro Pro Pro TyrGln Ser Asn Asn Asn Asn Asn Ser Glu Ile 540 545 550 AAA CCG CCG AGC TGCAAC AAC AAC AAC ATA CAG ATA AGC AAC AGC AAC 2812 Lys Pro Pro Ser Cys AsnAsn Asn Asn Ile Gln Ile Ser Asn Ser Asn 555 560 565 570 CTG GCG ACG ACACCA CCC ATT CCG CCT GCC AAA TAC AAT AAC AAC TCC 2860 Leu Ala Thr Thr ProPro Ile Pro Pro Ala Lys Tyr Asn Asn Asn Ser 575 580 585 TCC AAC ACG GGCGCG AAT AGC TCG GGC GGC AGC AAC GGA TCC ACC GGC 2908 Ser Asn Thr Gly AlaAsn Ser Ser Gly Gly Ser Asn Gly Ser Thr Gly 590 595 600 ACC ACC GCC TCCTCG TCG ACC AGC TGC AAG AAG ATC AAG CAC GCC TCG 2956 Thr Thr Ala Ser SerSer Thr Ser Cys Lys Lys Ile Lys His Ala Ser 605 610 615 CCC ATC CCG GAGCGC AAG AAG ATC TCC AAG GAG AAG GAG GAG GAG CGC 3004 Pro Ile Pro Glu ArgLys Lys Ile Ser Lys Glu Lys Glu Glu Glu Arg 620 625 630 AAG GAG TTC CGCATC AGG CAG TAC TCG CCG CAA GCC TTC AAG TTC TTC 3052 Lys Glu Phe Arg IleArg Gln Tyr Ser Pro Gln Ala Phe Lys Phe Phe 635 640 645 650 ATG GAG CAGCAC ATA GAG AAC GTG ATC AAG TCG TAT CGC CAG CGC ACG 3100 Met Glu Gln HisIle Glu Asn Val Ile Lys Ser Tyr Arg Gln Arg Thr 655 660 665 TAT CGC AAGAAT CAG CTG GAG AAG GAG ATG CAC AAA GTG GGA CTG CCC 3148 Tyr Arg Lys AsnGln Leu Glu Lys Glu Met His Lys Val Gly Leu Pro 670 675 680 GAT CAG ACCCAA ATC GAG ATG AGG AAA ATG CTG AAC CAA AAG GAG AGC 3196 Asp Gln Thr GlnIle Glu Met Arg Lys Met Leu Asn Gln Lys Glu Ser 685 690 695 AAC TAC ATTCGA TTG AAG CGC GCC AAG ATG GAC AAG AGC ATG TTC GTC 3244 Asn Tyr Ile ArgLeu Lys Arg Ala Lys Met Asp Lys Ser Met Phe Val 700 705 710 AAA CTG AAGCCC ATT GGA GTG GGT GCA TTT GGC GAG GTA ACG CTG GTG 3292 Lys Leu Lys ProIle Gly Val Gly Ala Phe Gly Glu Val Thr Leu Val 715 720 725 730 AGC AAAATC GAT ACC TCG AAC CAT TTG TAT GCG ATG AAA ACC CTG CGG 3340 Ser Lys IleAsp Thr Ser Asn His Leu Tyr Ala Met Lys Thr Leu Arg 735 740 745 AAA GCGGAC GTT CTC AAG CGG AAT CAG GTG GCA CAC GTG AAG GCC GAG 3388 Lys Ala AspVal Leu Lys Arg Asn Gln Val Ala His Val Lys Ala Glu 750 755 760 AGG GATATC CTC GCG GAA GCC GAC AAT AAC TGG GTG GTG AAG TTG TAC 3436 Arg Asp IleLeu Ala Glu Ala Asp Asn Asn Trp Val Val Lys Leu Tyr 765 770 775 TAC AGCTTC CAG GAC AAG GAT AAT CTG TAC TTT GTG ATG GAC TAC ATA 3484 Tyr Ser PheGln Asp Lys Asp Asn Leu Tyr Phe Val Met Asp Tyr Ile 780 785 790 CCA GGTGGT GAT CTG ATG TCG CTG CTC ATC AAA CTG GGC ATT TTC GAG 3532 Pro Gly GlyAsp Leu Met Ser Leu Leu Ile Lys Leu Gly Ile Phe Glu 795 800 805 810 GAGGAA CTG GCC AGA TTC TAC ATC GCC GAG GTC ACC TGC GCC GTG GAC 3580 Glu GluLeu Ala Arg Phe Tyr Ile Ala Glu Val Thr Cys Ala Val Asp 815 820 825 AGCGTT CAC AAA ATG GGC TTC ATT CAC AGA GAC ATC AAG CCT GAC AAC 3628 Ser ValHis Lys Met Gly Phe Ile His Arg Asp Ile Lys Pro Asp Asn 830 835 840 ATACTC ATC GAT AGG GAC GGA CAC ATA AAG CTC ACC GAC TTT GGC CTG 3676 Ile LeuIle Asp Arg Asp Gly His Ile Lys Leu Thr Asp Phe Gly Leu 845 850 855 TGCACG GGA TTC CGA TGG ACG CAC AAC TCG AAG TAC TAC CAG GAG AAC 3724 Cys ThrGly Phe Arg Trp Thr His Asn Ser Lys Tyr Tyr Gln Glu Asn 860 865 870 GGCAAT CAC TCG CGC CAG GAC TCG ATG GAG CCC TGG GAG GAA TAC TCC 3772 Gly AsnHis Ser Arg Gln Asp Ser Met Glu Pro Trp Glu Glu Tyr Ser 875 880 885 890GAG AAC GGA CCG AAG CCC ACC GTG CTG GAG AGG CGA CGG ATG CGC GAT 3820 GluAsn Gly Pro Lys Pro Thr Val Leu Glu Arg Arg Arg Met Arg Asp 895 900 905CAC CAA AGA GTC CTG GCC CAC TCG CTG GTG GGC ACC CCG AAC TAC ATA 3868 HisGln Arg Val Leu Ala His Ser Leu Val Gly Thr Pro Asn Tyr Ile 910 915 920GCT CCC GAG GTG CTG GAG AGG AGT GGG TAC ACG CAG CTG TGC GAC TAC 3916 AlaPro Glu Val Leu Glu Arg Ser Gly Tyr Thr Gln Leu Cys Asp Tyr 925 930 935TGG AGC GTG GGC GTC ATC CTT TAC GAG ATG CTG GTG GGT CAG CCG CCC 3964 TrpSer Val Gly Val Ile Leu Tyr Glu Met Leu Val Gly Gln Pro Pro 940 945 950TTT CTG GCC AAC AGT CCG CTG GAA ACG CAA CAA AAG GTC ATC AAC TGG 4012 PheLeu Ala Asn Ser Pro Leu Glu Thr Gln Gln Lys Val Ile Asn Trp 955 960 965970 GAG AAA ACG CTG CAT ATT CCG CCG CAG GCC GAG TTA TCC CGC GAG GCT 4060Glu Lys Thr Leu His Ile Pro Pro Gln Ala Glu Leu Ser Arg Glu Ala 975 980985 ACG GAC TTG ATA AGG AGG CTC TGT GCG TCG GCT GAC AAG CGG CTG GGC 4108Thr Asp Leu Ile Arg Arg Leu Cys Ala Ser Ala Asp Lys Arg Leu Gly 990 9951000 AAG AGC GTG GAC GAG GTC AAG AGC CAC GAC TTC TTC AAG GGC ATC GAC4156 Lys Ser Val Asp Glu Val Lys Ser His Asp Phe Phe Lys Gly Ile Asp1005 1010 1015 TTT GCG GAC ATG CGG AAG CAG AAA GCG CCC TAC ATA CCG GAAATC AAG 4204 Phe Ala Asp Met Arg Lys Gln Lys Ala Pro Tyr Ile Pro Glu IleLys 1020 1025 1030 CAC CCA ACG GAC ACA TCC AAC TTT GAT CCC GTG GAT CCGGAG AAG CTG 4252 His Pro Thr Asp Thr Ser Asn Phe Asp Pro Val Asp Pro GluLys Leu 1035 1040 1045 1050 CGC TCG AAT GAC TCC ACC ATG AGC AGC GGC GATGAT GTC GAC CAG AAT 4300 Arg Ser Asn Asp Ser Thr Met Ser Ser Gly Asp AspVal Asp Gln Asn 1055 1060 1065 GAC CGC ACT TTC CAC GGC TTT TTC GAA TTTACC TTC CGT CGC TTC TTC 4348 Asp Arg Thr Phe His Gly Phe Phe Glu Phe ThrPhe Arg Arg Phe Phe 1070 1075 1080 GAC GAC AAG CAG CCG CCG GAT ATG ACGGAC GAT CAG GCG CCG GTT TAC 4396 Asp Asp Lys Gln Pro Pro Asp Met Thr AspAsp Gln Ala Pro Val Tyr 1085 1090 1095 GTC TGA AATGGATGCT CTCCATGTGCCCAACACCAA CACCCCGCCC CCGAATCATT 4452 Val * 1100 GTTAGTCAAA TAGTCACAAAAAGGGGATAG AAACCATTGA GTGGGCTTGC ATTGTAAAGG 4512 AAGCGTGGCT ATAGAATGAAACTATCTATA TACATTATAT AAATTATAGG AGACAGTAGA 4572 GGCGGGAGCT ACGTATATACATACAAATAA TATACATATA TTTGATATAT ATATATATAT 4632 ATATGCCGTA GGGCATGAACTGAATAAATA TAAAACGGAG CCGAGTAGAG ATGAAACGAG 4692 AGGAGCGAGT CAGGACCTTCGACCTTTAAC TGAACATAGT ATATCCTTGT GCACTACTAC 4752 TCCACAACAA ATATATATTTTTAAATTGTT AGAATTCAAA AGGGACCAAC TGGAAATCGA 4812 ACCTTTCTGG TGCTCAAAGCAAAGCAAAGC AAAGCAAAAC AAAACGCCTT AAACTAAATG 4872 AGACGCGAAT TTACCCAACCACTTCACTCC TCTCCTTTCT CCACCTCCGA TCGGTGGCCG 4932 GATTCGAACT CAGCAGGCTGGTTGCATCCG GCCATCCCAT TGACTTCCCA TTCAGAATTG 4992 AGATTGCGAG GTGTGCGATGGAGAACGAAC GGAGACCAAA AGTCGCACGG CAGCGATATA 5052 AGCGGGTCTT ATAAGCCTAATCTAAATCTA AACTGGGAGA ACAGGACCTA TGTATGTCCT 5112 GCTATCCAAT TCGTCTATCACTGCTCTTCA TCTGTGTACG ACCCCCACCC CCCCCCTCCC 5172 CATCCAAAAG AACAAACTTAGACGTAGCCT ATGTGAAAAG CTAGCAATGT TAGACCAACT 5232 TGTTGAATGC CAAATGAAATTGTTTAGCCC CACGAGGAAA ACGCGGGGGA AATTCAACAC 5292 TTATTCTCTG ATAGCAAACGGAAAAGAAAG AAAGAAAAAA AAAAACAGAA ACAGTACGAG 5352 AAAATTGTAA TCTTCTTAATGTAATATTGT AAAGAACACG TTAATTGTAA TCTATGCTAG 5412 AGTTGTGTAG CGCCCTAAGATGTTTTTTAG TTTATAGACC GCTAACCGTA ATCTAGTTTA 5472 ATTCCTAACA CTAAGCGAGAGTACAGTACA TTGGTTTTTT TGTTTGTCGT AGGTTCGTTG 5532 GAAAATGCTT AACGGGAAACGATTTGTTTT TCTCTTTAAT TAGCTTCAGT TTGTATGTGC 5592 GTGTGTTTTT ATTATGACTTATATATAGTC CATCTGAATA TTCGTGGATG GAGCCTATTT 5652 TAAATGTGAG ATCGAGCTAATTGAAGGAAA TACAAACAAA CTCTGTGTGC CTTGGCCAAT 5712 TAGTTTAC 5720 1099amino acids amino acid unknown protein 2 Met His Pro Ala Gly Glu Lys ArgGly Gly Arg Pro Asn Asp Lys Tyr 1 5 10 15 Thr Ala Glu Ala Leu Glu SerIle Lys Gln Asp Leu Thr Arg Phe Glu 20 25 30 Val Gln Asn Asn His Arg AsnAsn Gln Asn Tyr Thr Pro Leu Arg Tyr 35 40 45 Thr Ala Thr Asn Gly Arg AsnAsp Ala Leu Thr Pro Asp Tyr His His 50 55 60 Ala Lys Gln Pro Met Glu ProPro Pro Ser Ala Ser Pro Ala Pro Asp 65 70 75 80 Val Val Ile Pro Pro ProPro Ala Ile Val Gly Gln Pro Gly Ala Gly 85 90 95 Ser Ile Ser Val Ser GlyVal Gly Val Gly Val Val Gly Val Ala Asn 100 105 110 Gly Arg Val Pro LysMet Met Thr Ala Leu Met Pro Asn Lys Leu Ile 115 120 125 Arg Lys Pro SerIle Glu Arg Asp Thr Ala Ser Ser His Tyr Leu Arg 130 135 140 Cys Ser ProAla Leu Asp Ser Gly Ala Gly Ser Ser Arg Ser Asp Ser 145 150 155 160 ProHis Ser His His Thr His Gln Pro Ser Ser Arg Thr Val Gly Asn 165 170 175Pro Gly Gly Asn Gly Gly Phe Ser Pro Ser Pro Ser Gly Phe Ser Glu 180 185190 Val Ala Pro Pro Ala Pro Pro Pro Arg Asn Pro Thr Ala Ser Ser Ala 195200 205 Ala Thr Pro Pro Pro Pro Val Pro Pro Thr Ser Gln Ala Tyr Val Lys210 215 220 Arg Arg Ser Pro Ala Leu Asn Asn Arg Pro Pro Ala Ile Ala ProPro 225 230 235 240 Thr Gln Arg Gly Asn Ser Pro Val Ile Thr Gln Asn GlyLeu Lys Asn 245 250 255 Pro Gln Gln Gln Leu Thr Gln Gln Leu Lys Ser LeuAsn Leu Tyr Pro 260 265 270 Gly Gly Gly Ser Gly Ala Val Val Glu Pro ProPro Pro Tyr Leu Ile 275 280 285 Gln Gly Gly Ala Gly Gly Ala Ala Pro ProPro Pro Pro Pro Ser Tyr 290 295 300 Thr Ala Ser Met Gln Ser Arg Gln SerPro Thr Gln Ser Gln Gln Ser 305 310 315 320 Asp Tyr Arg Lys Ser Pro SerSer Gly Ile Tyr Ser Ala Thr Ser Ala 325 330 335 Gly Ser Pro Ser Pro IleThr Val Ser Leu Pro Pro Ala Pro Leu Ala 340 345 350 Lys Pro Gln Pro ArgVal Tyr Gln Ala Arg Ser Gln Gln Pro Ile Ile 355 360 365 Met Gln Ser ValLys Ser Thr Gln Val Gln Lys Pro Val Leu Gln Thr 370 375 380 Ala Val AlaArg Gln Ser Pro Ser Ser Ala Ser Ala Ser Asn Ser Pro 385 390 395 400 ValHis Val Leu Ala Ala Pro Pro Ser Tyr Pro Gln Lys Ser Ala Ala 405 410 415Val Val Gln Gln Gln Gln Gln Ala Ala Ala Ala Ala His Gln Gln Gln 420 425430 His Gln His Gln Gln Ser Lys Pro Pro Thr Pro Thr Thr Pro Pro Leu 435440 445 Val Gly Leu Asn Ser Lys Pro Asn Cys Leu Glu Pro Pro Ser Tyr Ala450 455 460 Lys Ser Met Gln Ala Lys Ala Ala Thr Val Val Gln Gln Gln GlnGln 465 470 475 480 Gln Gln Gln Gln Gln Gln Val Gln Gln Gln Gln Val GlnGln Gln Gln 485 490 495 Gln Gln Gln Gln Gln Gln Leu Gln Ala Leu Arg ValLeu Gln Ala Gln 500 505 510 Ala Gln Arg Glu Arg Asp Gln Arg Glu Arg GluArg Asp Gln Gln Lys 515 520 525 Leu Ala Asn Gly Asn Pro Gly Arg Gln MetLeu Pro Pro Pro Pro Tyr 530 535 540 Gln Ser Asn Asn Asn Asn Asn Ser GluIle Lys Pro Pro Ser Cys Asn 545 550 555 560 Asn Asn Asn Ile Gln Ile SerAsn Ser Asn Leu Ala Thr Thr Pro Pro 565 570 575 Ile Pro Pro Ala Lys TyrAsn Asn Asn Ser Ser Asn Thr Gly Ala Asn 580 585 590 Ser Ser Gly Gly SerAsn Gly Ser Thr Gly Thr Thr Ala Ser Ser Ser 595 600 605 Thr Ser Cys LysLys Ile Lys His Ala Ser Pro Ile Pro Glu Arg Lys 610 615 620 Lys Ile SerLys Glu Lys Glu Glu Glu Arg Lys Glu Phe Arg Ile Arg 625 630 635 640 GlnTyr Ser Pro Gln Ala Phe Lys Phe Phe Met Glu Gln His Ile Glu 645 650 655Asn Val Ile Lys Ser Tyr Arg Gln Arg Thr Tyr Arg Lys Asn Gln Leu 660 665670 Glu Lys Glu Met His Lys Val Gly Leu Pro Asp Gln Thr Gln Ile Glu 675680 685 Met Arg Lys Met Leu Asn Gln Lys Glu Ser Asn Tyr Ile Arg Leu Lys690 695 700 Arg Ala Lys Met Asp Lys Ser Met Phe Val Lys Leu Lys Pro IleGly 705 710 715 720 Val Gly Ala Phe Gly Glu Val Thr Leu Val Ser Lys IleAsp Thr Ser 725 730 735 Asn His Leu Tyr Ala Met Lys Thr Leu Arg Lys AlaAsp Val Leu Lys 740 745 750 Arg Asn Gln Val Ala His Val Lys Ala Glu ArgAsp Ile Leu Ala Glu 755 760 765 Ala Asp Asn Asn Trp Val Val Lys Leu TyrTyr Ser Phe Gln Asp Lys 770 775 780 Asp Asn Leu Tyr Phe Val Met Asp TyrIle Pro Gly Gly Asp Leu Met 785 790 795 800 Ser Leu Leu Ile Lys Leu GlyIle Phe Glu Glu Glu Leu Ala Arg Phe 805 810 815 Tyr Ile Ala Glu Val ThrCys Ala Val Asp Ser Val His Lys Met Gly 820 825 830 Phe Ile His Arg AspIle Lys Pro Asp Asn Ile Leu Ile Asp Arg Asp 835 840 845 Gly His Ile LysLeu Thr Asp Phe Gly Leu Cys Thr Gly Phe Arg Trp 850 855 860 Thr His AsnSer Lys Tyr Tyr Gln Glu Asn Gly Asn His Ser Arg Gln 865 870 875 880 AspSer Met Glu Pro Trp Glu Glu Tyr Ser Glu Asn Gly Pro Lys Pro 885 890 895Thr Val Leu Glu Arg Arg Arg Met Arg Asp His Gln Arg Val Leu Ala 900 905910 His Ser Leu Val Gly Thr Pro Asn Tyr Ile Ala Pro Glu Val Leu Glu 915920 925 Arg Ser Gly Tyr Thr Gln Leu Cys Asp Tyr Trp Ser Val Gly Val Ile930 935 940 Leu Tyr Glu Met Leu Val Gly Gln Pro Pro Phe Leu Ala Asn SerPro 945 950 955 960 Leu Glu Thr Gln Gln Lys Val Ile Asn Trp Glu Lys ThrLeu His Ile 965 970 975 Pro Pro Gln Ala Glu Leu Ser Arg Glu Ala Thr AspLeu Ile Arg Arg 980 985 990 Leu Cys Ala Ser Ala Asp Lys Arg Leu Gly LysSer Val Asp Glu Val 995 1000 1005 Lys Ser His Asp Phe Phe Lys Gly IleAsp Phe Ala Asp Met Arg Lys 1010 1015 1020 Gln Lys Ala Pro Tyr Ile ProGlu Ile Lys His Pro Thr Asp Thr Ser 1025 1030 1035 1040 Asn Phe Asp ProVal Asp Pro Glu Lys Leu Arg Ser Asn Asp Ser Thr 1045 1050 1055 Met SerSer Gly Asp Asp Val Asp Gln Asn Asp Arg Thr Phe His Gly 1060 1065 1070Phe Phe Glu Phe Thr Phe Arg Arg Phe Phe Asp Asp Lys Gln Pro Pro 10751080 1085 Asp Met Thr Asp Asp Gln Ala Pro Val Tyr Val 1090 1095 11003983 amino acids nucleic acid double unknown cDNA CDS 231..3623 3ACCTTTGGGT TGCTGGGACG GACTCTGGCC GCCTCAGCGT CCGCCCTCAG GCCCGTGGCC 60GCTGTCCAGG AGCTCTGCTC TCCCCTCCAG AGTTAATTAT TTATATTGTA AAGAATTTTA 120ACAGTCCTGG GGACTTCCTT GAAGGATCAT TTTCACTTTT GCTCAGAAGA AAGCTCTGGA 180TCTATCAAAT AAAGAAGTCC TTCGTGTGGG CTACATATAT AGATGTTTTC ATG AAG 236 MetLys 1 AGG AGT GAA AAG CCA GAA GGA TAT AGA CAA ATG AGG CCT AAG ACC TTT284 Arg Ser Glu Lys Pro Glu Gly Tyr Arg Gln Met Arg Pro Lys Thr Phe 5 1015 CCT GCC AGT AAC TAT ACT GTC AGT AGC CGG CAA ATG TTA CAA GAA ATT 332Pro Ala Ser Asn Tyr Thr Val Ser Ser Arg Gln Met Leu Gln Glu Ile 20 25 30CGG GAA TCC CTT AGG AAT TTA TCT AAA CCA TCT GAT GCT GCT AAG GCT 380 ArgGlu Ser Leu Arg Asn Leu Ser Lys Pro Ser Asp Ala Ala Lys Ala 35 40 45 50GAG CAT AAC ATG AGT AAA ATG TCA ACC GAA GAT CCT CGA CAA GTC AGA 428 GluHis Asn Met Ser Lys Met Ser Thr Glu Asp Pro Arg Gln Val Arg 55 60 65 AATCCA CCC AAA TTT GGG ACG CAT CAT AAA GCC TTG CAG GAA ATT CGA 476 Asn ProPro Lys Phe Gly Thr His His Lys Ala Leu Gln Glu Ile Arg 70 75 80 AAC TCTCTG CTT CCA TTT GCA AAT GAA ACA AAT TCT TCT CGG AGT ACT 524 Asn Ser LeuLeu Pro Phe Ala Asn Glu Thr Asn Ser Ser Arg Ser Thr 85 90 95 TCA GAA GTTAAT CCA CAA ATG CTT CAA GAC TTG CAA GCT GCT GGA TTT 572 Ser Glu Val AsnPro Gln Met Leu Gln Asp Leu Gln Ala Ala Gly Phe 100 105 110 GAT GAG GATATG GTT ATA CAA GCT CTT CAG AAA ACT AAC AAC AGA AGT 620 Asp Glu Asp MetVal Ile Gln Ala Leu Gln Lys Thr Asn Asn Arg Ser 115 120 125 130 ATA GAAGCA GCA ATT GAA TTC ATT AGT AAA ATG AGT TAC CAA GAT CCT 668 Ile Glu AlaAla Ile Glu Phe Ile Ser Lys Met Ser Tyr Gln Asp Pro 135 140 145 CGA CGAGAG CAG ATG GCT GCA GCA GCT GCC AGA CCT ATT AAT GCC AGC 716 Arg Arg GluGln Met Ala Ala Ala Ala Ala Arg Pro Ile Asn Ala Ser 150 155 160 ATG AAACCA GGG AAT GTG CAG CAA TCA GTT AAC CGC AAA CAG AGC TGG 764 Met Lys ProGly Asn Val Gln Gln Ser Val Asn Arg Lys Gln Ser Trp 165 170 175 AAA GGTTCT AAA GAA TCC TTA GTT CCT CAG AGG CAT GGC CCG CCA CTA 812 Lys Gly SerLys Glu Ser Leu Val Pro Gln Arg His Gly Pro Pro Leu 180 185 190 GGA GAAAGT GTG GCC TAT CAT TCT GAG AGT CCC AAC TCA CAG ACA GAT 860 Gly Glu SerVal Ala Tyr His Ser Glu Ser Pro Asn Ser Gln Thr Asp 195 200 205 210 GTAGGA AGA CCT TTG TCT GGA TCT GGT ATA TCA GCA TTT GTT CAA GCT 908 Val GlyArg Pro Leu Ser Gly Ser Gly Ile Ser Ala Phe Val Gln Ala 215 220 225 CACCCT AGC AAC GGA CAG AGA GTG AAC CCC CCA CCA CCA CCT CAA GTA 956 His ProSer Asn Gly Gln Arg Val Asn Pro Pro Pro Pro Pro Gln Val 230 235 240 AGGAGT GTT ACT CCT CCA CCA CCT CCA AGA GGC CAG ACT CCC CCT CCA 1004 Arg SerVal Thr Pro Pro Pro Pro Pro Arg Gly Gln Thr Pro Pro Pro 245 250 255 AGAGGT ACA ACT CCA CCT CCC CCT TCA TGG GAA CCA AAC TCT CAA ACA 1052 Arg GlyThr Thr Pro Pro Pro Pro Ser Trp Glu Pro Asn Ser Gln Thr 260 265 270 AAGCGC TAT TCT GGA AAC ATG GAA TAC GTA ATC TCC CGA ATC TCT CCT 1100 Lys ArgTyr Ser Gly Asn Met Glu Tyr Val Ile Ser Arg Ile Ser Pro 275 280 285 290GTC CCA CCT GGG GCA TGG CAA GAG GGC TAT CCT CCA CCA CCT CTC AAC 1148 ValPro Pro Gly Ala Trp Gln Glu Gly Tyr Pro Pro Pro Pro Leu Asn 295 300 305ACT TCC CCC ATG AAT CCT CCT AAT CAA GGA CAG AGA GGC ATT AGT TCT 1196 ThrSer Pro Met Asn Pro Pro Asn Gln Gly Gln Arg Gly Ile Ser Ser 310 315 320GTT CCT GTT GGC AGA CAA CCA ATC ATC ATG CAG AGT TCT AGC AAA TTT 1244 ValPro Val Gly Arg Gln Pro Ile Ile Met Gln Ser Ser Ser Lys Phe 325 330 335AAC TTT CCA TCA GGG AGA CCT GGA ATG CAG AAT GGT ACT GGA CAA ACT 1292 AsnPhe Pro Ser Gly Arg Pro Gly Met Gln Asn Gly Thr Gly Gln Thr 340 345 350GAT TTC ATG ATA CAC CAA AAT GTT GTC CCT GCT GGC ACT GTG AAT CGG 1340 AspPhe Met Ile His Gln Asn Val Val Pro Ala Gly Thr Val Asn Arg 355 360 365370 CAG CCA CCA CCT CCA TAT CCT CTG ACA GCA GCT AAT GGA CAA AGC CCT 1388Gln Pro Pro Pro Pro Tyr Pro Leu Thr Ala Ala Asn Gly Gln Ser Pro 375 380385 TCT GCT TTA CAA ACA GGG GGA TCT GCT GCT CCT TCG TCA TAT ACA AAT 1436Ser Ala Leu Gln Thr Gly Gly Ser Ala Ala Pro Ser Ser Tyr Thr Asn 390 395400 GGA AGT ATT CCT CAG TCT ATG ATG GTG CCA AAC AGA AAT AGT CAT AAC 1484Gly Ser Ile Pro Gln Ser Met Met Val Pro Asn Arg Asn Ser His Asn 405 410415 ATG GAA CTA TAT AAC ATT AGT GTA CCT GGA CTG CAA ACA AAT TGG CCT 1532Met Glu Leu Tyr Asn Ile Ser Val Pro Gly Leu Gln Thr Asn Trp Pro 420 425430 CAG TCA TCT TCT GCT CCA GCC CAG TCA TCC CCG AGC AGT GGG CAT GAA 1580Gln Ser Ser Ser Ala Pro Ala Gln Ser Ser Pro Ser Ser Gly His Glu 435 440445 450 ATC CCT ACA TGG CAA CCT AAC ATA CCA GTG AGG TCA AAT TCT TTT AAT1628 Ile Pro Thr Trp Gln Pro Asn Ile Pro Val Arg Ser Asn Ser Phe Asn 455460 465 AAC CCA TTA GGA AAT AGA GCA AGT CAC TCT GCT AAT TCT CAG CCT TCT1676 Asn Pro Leu Gly Asn Arg Ala Ser His Ser Ala Asn Ser Gln Pro Ser 470475 480 GCT ACA ACA GTC ACT GCA ATT ACA CCA GCT CCT ATT CAA CAG CCT GTG1724 Ala Thr Thr Val Thr Ala Ile Thr Pro Ala Pro Ile Gln Gln Pro Val 485490 495 AAA AGT ATG CGT GTA TTA AAA CCA GAG CTA CAG ACT GCT TTA GCA CCT1772 Lys Ser Met Arg Val Leu Lys Pro Glu Leu Gln Thr Ala Leu Ala Pro 500505 510 ACA CAC CCT TCT TGG ATA CCA CAG CCA ATT CAA ACT GTT CAA CCC AGT1820 Thr His Pro Ser Trp Ile Pro Gln Pro Ile Gln Thr Val Gln Pro Ser 515520 525 530 CCT TTT CCT GAG GGA ACC GCT TCA AAT GTG ACT GTG ATG CCA CCTGTT 1868 Pro Phe Pro Glu Gly Thr Ala Ser Asn Val Thr Val Met Pro Pro Val535 540 545 GCT GAA GCT CCA AAC TAT CAA GGA CCA CCA CCA CCC TAC CCA AAACAT 1916 Ala Glu Ala Pro Asn Tyr Gln Gly Pro Pro Pro Pro Tyr Pro Lys His550 555 560 CTG CTG CAC CAA AAC CCA TCT GTT CCT CCA TAC GAG TCA ATC AGTAAG 1964 Leu Leu His Gln Asn Pro Ser Val Pro Pro Tyr Glu Ser Ile Ser Lys565 570 575 CCT AGC AAA GAG GAT CAG CCA AGC TTG CCC AAG GAA GAT GAG AGTGAA 2012 Pro Ser Lys Glu Asp Gln Pro Ser Leu Pro Lys Glu Asp Glu Ser Glu580 585 590 AAG AGT TAT GAA AAT GTT GAT AGT GGG GAT AAA GAA AAG AAA CAGATT 2060 Lys Ser Tyr Glu Asn Val Asp Ser Gly Asp Lys Glu Lys Lys Gln Ile595 600 605 610 ACA ACT TCA CCT ATT ACT GTT AGG AAA AAC AAG AAA GAT GAAGAG CGA 2108 Thr Thr Ser Pro Ile Thr Val Arg Lys Asn Lys Lys Asp Glu GluArg 615 620 625 AGG GAA TCT CGT ATT CAA AGT TAT TCT CCT CAA GCA TTT AAATTC TTT 2156 Arg Glu Ser Arg Ile Gln Ser Tyr Ser Pro Gln Ala Phe Lys PhePhe 630 635 640 ATG GAG CAA CAT GTA GAA AAT GTA CTC AAA TCT CAT CAG CAGCGT CTA 2204 Met Glu Gln His Val Glu Asn Val Leu Lys Ser His Gln Gln ArgLeu 645 650 655 CAT CGT AAA AAA CAA TTA GAG AAT GAA ATG ATG CGG GTT GGATTA TCT 2252 His Arg Lys Lys Gln Leu Glu Asn Glu Met Met Arg Val Gly LeuSer 660 665 670 CAA GAT GCC CAG GAT CAA ATG AGA AAG ATG CTT TGC CAA AAAGAA TCT 2300 Gln Asp Ala Gln Asp Gln Met Arg Lys Met Leu Cys Gln Lys GluSer 675 680 685 690 AAT TAC ATC CGT CTT AAA AGG GCT AAA ATG GAC AAG TCTATG TTT GTG 2348 Asn Tyr Ile Arg Leu Lys Arg Ala Lys Met Asp Lys Ser MetPhe Val 695 700 705 AAG ATA AAG ACA CTA GGA ATA GGA GCA TTT GGT GAA GTCTGT CTA GCA 2396 Lys Ile Lys Thr Leu Gly Ile Gly Ala Phe Gly Glu Val CysLeu Ala 710 715 720 AGA AAA GTA GAT ACT AAG GCT TTG TAT GCA ACA AAA ACTCTT CGA AAG 2444 Arg Lys Val Asp Thr Lys Ala Leu Tyr Ala Thr Lys Thr LeuArg Lys 725 730 735 AAA GAT GTT CTT CTT CGA AAT CAA GTC GCT CAT GTT AAGGCT GAG AGA 2492 Lys Asp Val Leu Leu Arg Asn Gln Val Ala His Val Lys AlaGlu Arg 740 745 750 GAT ATC CTG GCT GAA GCT GAC AAT GAA TGG GTA GTT CGTCTA TAT TAT 2540 Asp Ile Leu Ala Glu Ala Asp Asn Glu Trp Val Val Arg LeuTyr Tyr 755 760 765 770 TCA TTC CAA GAT AAG GAC AAT TTA TAC TTT GTA ATGGAC TAC ATT CCT 2588 Ser Phe Gln Asp Lys Asp Asn Leu Tyr Phe Val Met AspTyr Ile Pro 775 780 785 GGG GGT GAT ATG ATG AGC CTA TTA ATT AGA ATG GGCATC TTT CCA GAA 2636 Gly Gly Asp Met Met Ser Leu Leu Ile Arg Met Gly IlePhe Pro Glu 790 795 800 AGT CTG GCA CGA TTC TAC ATA GCA GAA CTT ACC TGTGCA GTT GAA AGT 2684 Ser Leu Ala Arg Phe Tyr Ile Ala Glu Leu Thr Cys AlaVal Glu Ser 805 810 815 GTT CAT AAA ATG GGT TTT ATT CAT AGA GAT ATT AAACCT GAT AAT ATT 2732 Val His Lys Met Gly Phe Ile His Arg Asp Ile Lys ProAsp Asn Ile 820 825 830 TTG ATT GAT CGT GAT GGT CAT ATT AAA TTG ACT GACTTT GGC CTC TGC 2780 Leu Ile Asp Arg Asp Gly His Ile Lys Leu Thr Asp PheGly Leu Cys 835 840 845 850 ACT GGC TTC AGA TGG ACA CAC GAT TCT AAG TACTAT CAG AGT GGT GAC 2828 Thr Gly Phe Arg Trp Thr His Asp Ser Lys Tyr TyrGln Ser Gly Asp 855 860 865 CAT CCA CGG CAA GAT AGC ATG GAT TTC AGT AATGAA TGG GGG GAT CCC 2876 His Pro Arg Gln Asp Ser Met Asp Phe Ser Asn GluTrp Gly Asp Pro 870 875 880 TCA AGC TGT CGA TGT GGA GAC AGA CTG AAG CCATTA GAG CGG AGA GCT 2924 Ser Ser Cys Arg Cys Gly Asp Arg Leu Lys Pro LeuGlu Arg Arg Ala 885 890 895 GCA CGC CAG CAC CAG CGA TGT CTA GCA CAT TCTTTG GTT GGG ACT CCC 2972 Ala Arg Gln His Gln Arg Cys Leu Ala His Ser LeuVal Gly Thr Pro 900 905 910 AAT TAT ATT GCA CCT GAA GTG TTG CTA CGA ACAGGA TAC ACA CAG TTG 3020 Asn Tyr Ile Ala Pro Glu Val Leu Leu Arg Thr GlyTyr Thr Gln Leu 915 920 925 930 TGT GAT TGG TGG AGT GTT GGT GTT ATT CTTTTT GAA ATG TTG GTG GGA 3068 Cys Asp Trp Trp Ser Val Gly Val Ile Leu PheGlu Met Leu Val Gly 935 940 945 CAA CCT CCT TTC TTG GCA CAA ACA CCA TTAGAA ACA CAA ATG AAG GTT 3116 Gln Pro Pro Phe Leu Ala Gln Thr Pro Leu GluThr Gln Met Lys Val 950 955 960 ATC AAC TGG CAA ACA TCT CTT CAC ATT CCACCA CAA GCT AAA CTC AGT 3164 Ile Asn Trp Gln Thr Ser Leu His Ile Pro ProGln Ala Lys Leu Ser 965 970 975 CCT GAA GCT TCT GAT CTT ATT ATT AAA CTTTGC CGA GGA CCC GAA GAT 3212 Pro Glu Ala Ser Asp Leu Ile Ile Lys Leu CysArg Gly Pro Glu Asp 980 985 990 CGC TTA GGC AAG AAT GGT GCT GAT GAA ATAAAA GCT CAT CCA TTT TTT 3260 Arg Leu Gly Lys Asn Gly Ala Asp Glu Ile LysAla His Pro Phe Phe 995 1000 1005 1010 AAA ACA ATT GAC TTC TCC AGT GACCTG AGA CAG CAG TCT GCT TCA TAC 3308 Lys Thr Ile Asp Phe Ser Ser Asp LeuArg Gln Gln Ser Ala Ser Tyr 1015 1020 1025 ATT CCT AAA ATC ACA CAC CCAACA GAT ACA TCA AAT TTT GAT CCT GTT 3356 Ile Pro Lys Ile Thr His Pro ThrAsp Thr Ser Asn Phe Asp Pro Val 1030 1035 1040 GAT CCT GAT AAA TTA TGGAGT GAT GAT AAC GAG GAA GAA AAT GTA AAT 3404 Asp Pro Asp Lys Leu Trp SerAsp Asp Asn Glu Glu Glu Asn Val Asn 1045 1050 1055 GAC ACT CTC AAT GGATGG TAT AAA AAT GGA AAG CAT CCT GAA CAT GCA 3452 Asp Thr Leu Asn Gly TrpTyr Lys Asn Gly Lys His Pro Glu His Ala 1060 1065 1070 TTC TAT GAA TTTACC TTC CGA AGG TTT TTT GAT GAC AAT GGC TAC CCA 3500 Phe Tyr Glu Phe ThrPhe Arg Arg Phe Phe Asp Asp Asn Gly Tyr Pro 1075 1080 1085 1090 TAT AATTAT CCG AAG CCT ATT GAA TAT GAA TAC ATT AAT TCA CAA GGC 3548 Tyr Asn TyrPro Lys Pro Ile Glu Tyr Glu Tyr Ile Asn Ser Gln Gly 1095 1100 1105 TCAGAG CAG CAG TCG GAT GAA GAT GAT CAA AAC ACA GGC TCA GAG ATT 3596 Ser GluGln Gln Ser Asp Glu Asp Asp Gln Asn Thr Gly Ser Glu Ile 1110 1115 1120AAA AAT CGC GAT CTA GTA TAT GTT TAA CACACTAGTA AATAAATGTA 3643 Lys AsnArg Asp Leu Val Tyr Val 1125 1130 ATGAGGATTT GTAAAAGGGC CTGAAATGCGAGGTGTTTTG AGGTTCTGAG AGTAAAATTA 3703 TGCAAATATG ACAGAGCTAT ATATGTGTGCTCTGTGTACA ATATTTTATT TTCCTAAATT 3763 ATGGGAAATC CTTTTAAAAT GTTAATTTATTCCAGCCGTT TAAATCAGTA TTTAGAAAAA 3823 AATTGTTATA AGGAAAGTAA ATTATGAACTGAATATTATA GTCAGTTCTT GGTACTTAAA 3883 GTACTTAAAA TAAGTAGTGC TTTGTTTAAAAGGAGAAACC TGGTATCTAT TTGTATATAT 3943 GCTAAATAAT TTTAAAATAC AAGAGTTTTTGAAATTTTTT T 3984 1130 amino acids amino acid unknown protein 4 Met LysArg Ser Glu Lys Pro Glu Gly Tyr Arg Gln Met Arg Pro Lys 1 5 10 15 ThrPhe Pro Ala Ser Asn Tyr Thr Val Ser Ser Arg Gln Met Leu Gln 20 25 30 GluIle Arg Glu Ser Leu Arg Asn Leu Ser Lys Pro Ser Asp Ala Ala 35 40 45 LysAla Glu His Asn Met Ser Lys Met Ser Thr Glu Asp Pro Arg Gln 50 55 60 ValArg Asn Pro Pro Lys Phe Gly Thr His His Lys Ala Leu Gln Glu 65 70 75 80Ile Arg Asn Ser Leu Leu Pro Phe Ala Asn Glu Thr Asn Ser Ser Arg 85 90 95Ser Thr Ser Glu Val Asn Pro Gln Met Leu Gln Asp Leu Gln Ala Ala 100 105110 Gly Phe Asp Glu Asp Met Val Ile Gln Ala Leu Gln Lys Thr Asn Asn 115120 125 Arg Ser Ile Glu Ala Ala Ile Glu Phe Ile Ser Lys Met Ser Tyr Gln130 135 140 Asp Pro Arg Arg Glu Gln Met Ala Ala Ala Ala Ala Arg Pro IleAsn 145 150 155 160 Ala Ser Met Lys Pro Gly Asn Val Gln Gln Ser Val AsnArg Lys Gln 165 170 175 Ser Trp Lys Gly Ser Lys Glu Ser Leu Val Pro GlnArg His Gly Pro 180 185 190 Pro Leu Gly Glu Ser Val Ala Tyr His Ser GluSer Pro Asn Ser Gln 195 200 205 Thr Asp Val Gly Arg Pro Leu Ser Gly SerGly Ile Ser Ala Phe Val 210 215 220 Gln Ala His Pro Ser Asn Gly Gln ArgVal Asn Pro Pro Pro Pro Pro 225 230 235 240 Gln Val Arg Ser Val Thr ProPro Pro Pro Pro Arg Gly Gln Thr Pro 245 250 255 Pro Pro Arg Gly Thr ThrPro Pro Pro Pro Ser Trp Glu Pro Asn Ser 260 265 270 Gln Thr Lys Arg TyrSer Gly Asn Met Glu Tyr Val Ile Ser Arg Ile 275 280 285 Ser Pro Val ProPro Gly Ala Trp Gln Glu Gly Tyr Pro Pro Pro Pro 290 295 300 Leu Asn ThrSer Pro Met Asn Pro Pro Asn Gln Gly Gln Arg Gly Ile 305 310 315 320 SerSer Val Pro Val Gly Arg Gln Pro Ile Ile Met Gln Ser Ser Ser 325 330 335Lys Phe Asn Phe Pro Ser Gly Arg Pro Gly Met Gln Asn Gly Thr Gly 340 345350 Gln Thr Asp Phe Met Ile His Gln Asn Val Val Pro Ala Gly Thr Val 355360 365 Asn Arg Gln Pro Pro Pro Pro Tyr Pro Leu Thr Ala Ala Asn Gly Gln370 375 380 Ser Pro Ser Ala Leu Gln Thr Gly Gly Ser Ala Ala Pro Ser SerTyr 385 390 395 400 Thr Asn Gly Ser Ile Pro Gln Ser Met Met Val Pro AsnArg Asn Ser 405 410 415 His Asn Met Glu Leu Tyr Asn Ile Ser Val Pro GlyLeu Gln Thr Asn 420 425 430 Trp Pro Gln Ser Ser Ser Ala Pro Ala Gln SerSer Pro Ser Ser Gly 435 440 445 His Glu Ile Pro Thr Trp Gln Pro Asn IlePro Val Arg Ser Asn Ser 450 455 460 Phe Asn Asn Pro Leu Gly Asn Arg AlaSer His Ser Ala Asn Ser Gln 465 470 475 480 Pro Ser Ala Thr Thr Val ThrAla Ile Thr Pro Ala Pro Ile Gln Gln 485 490 495 Pro Val Lys Ser Met ArgVal Leu Lys Pro Glu Leu Gln Thr Ala Leu 500 505 510 Ala Pro Thr His ProSer Trp Ile Pro Gln Pro Ile Gln Thr Val Gln 515 520 525 Pro Ser Pro PhePro Glu Gly Thr Ala Ser Asn Val Thr Val Met Pro 530 535 540 Pro Val AlaGlu Ala Pro Asn Tyr Gln Gly Pro Pro Pro Pro Tyr Pro 545 550 555 560 LysHis Leu Leu His Gln Asn Pro Ser Val Pro Pro Tyr Glu Ser Ile 565 570 575Ser Lys Pro Ser Lys Glu Asp Gln Pro Ser Leu Pro Lys Glu Asp Glu 580 585590 Ser Glu Lys Ser Tyr Glu Asn Val Asp Ser Gly Asp Lys Glu Lys Lys 595600 605 Gln Ile Thr Thr Ser Pro Ile Thr Val Arg Lys Asn Lys Lys Asp Glu610 615 620 Glu Arg Arg Glu Ser Arg Ile Gln Ser Tyr Ser Pro Gln Ala PheLys 625 630 635 640 Phe Phe Met Glu Gln His Val Glu Asn Val Leu Lys SerHis Gln Gln 645 650 655 Arg Leu His Arg Lys Lys Gln Leu Glu Asn Glu MetMet Arg Val Gly 660 665 670 Leu Ser Gln Asp Ala Gln Asp Gln Met Arg LysMet Leu Cys Gln Lys 675 680 685 Glu Ser Asn Tyr Ile Arg Leu Lys Arg AlaLys Met Asp Lys Ser Met 690 695 700 Phe Val Lys Ile Lys Thr Leu Gly IleGly Ala Phe Gly Glu Val Cys 705 710 715 720 Leu Ala Arg Lys Val Asp ThrLys Ala Leu Tyr Ala Thr Lys Thr Leu 725 730 735 Arg Lys Lys Asp Val LeuLeu Arg Asn Gln Val Ala His Val Lys Ala 740 745 750 Glu Arg Asp Ile LeuAla Glu Ala Asp Asn Glu Trp Val Val Arg Leu 755 760 765 Tyr Tyr Ser PheGln Asp Lys Asp Asn Leu Tyr Phe Val Met Asp Tyr 770 775 780 Ile Pro GlyGly Asp Met Met Ser Leu Leu Ile Arg Met Gly Ile Phe 785 790 795 800 ProGlu Ser Leu Ala Arg Phe Tyr Ile Ala Glu Leu Thr Cys Ala Val 805 810 815Glu Ser Val His Lys Met Gly Phe Ile His Arg Asp Ile Lys Pro Asp 820 825830 Asn Ile Leu Ile Asp Arg Asp Gly His Ile Lys Leu Thr Asp Phe Gly 835840 845 Leu Cys Thr Gly Phe Arg Trp Thr His Asp Ser Lys Tyr Tyr Gln Ser850 855 860 Gly Asp His Pro Arg Gln Asp Ser Met Asp Phe Ser Asn Glu TrpGly 865 870 875 880 Asp Pro Ser Ser Cys Arg Cys Gly Asp Arg Leu Lys ProLeu Glu Arg 885 890 895 Arg Ala Ala Arg Gln His Gln Arg Cys Leu Ala HisSer Leu Val Gly 900 905 910 Thr Pro Asn Tyr Ile Ala Pro Glu Val Leu LeuArg Thr Gly Tyr Thr 915 920 925 Gln Leu Cys Asp Trp Trp Ser Val Gly ValIle Leu Phe Glu Met Leu 930 935 940 Val Gly Gln Pro Pro Phe Leu Ala GlnThr Pro Leu Glu Thr Gln Met 945 950 955 960 Lys Val Ile Asn Trp Gln ThrSer Leu His Ile Pro Pro Gln Ala Lys 965 970 975 Leu Ser Pro Glu Ala SerAsp Leu Ile Ile Lys Leu Cys Arg Gly Pro 980 985 990 Glu Asp Arg Leu GlyLys Asn Gly Ala Asp Glu Ile Lys Ala His Pro 995 1000 1005 Phe Phe LysThr Ile Asp Phe Ser Ser Asp Leu Arg Gln Gln Ser Ala 1010 1015 1020 SerTyr Ile Pro Lys Ile Thr His Pro Thr Asp Thr Ser Asn Phe Asp 1025 10301035 1040 Pro Val Asp Pro Asp Lys Leu Trp Ser Asp Asp Asn Glu Glu GluAsn 1045 1050 1055 Val Asn Asp Thr Leu Asn Gly Trp Tyr Lys Asn Gly LysHis Pro Glu 1060 1065 1070 His Ala Phe Tyr Glu Phe Thr Phe Arg Arg PhePhe Asp Asp Asn Gly 1075 1080 1085 Tyr Pro Tyr Asn Tyr Pro Lys Pro IleGlu Tyr Glu Tyr Ile Asn Ser 1090 1095 1100 Gln Gly Ser Glu Gln Gln SerAsp Glu Asp Asp Gln Asn Thr Gly Ser 1105 1110 1115 1120 Glu Ile Lys AsnArg Asp Leu Val Tyr Val 1125 1130 3212 amino acids nucleic acid doubleunknown cDNA CDS 1..2889 5 GTG CAA CAT TCA ATT AAC CGA AAA CAA AGC TGGAAA GGT TCT AAA GAG 48 Val Gln His Ser Ile Asn Arg Lys Gln Ser Trp LysGly Ser Lys Glu 1 5 10 15 TCT CTA GTT CCT CAG AGA CAC GGC CCA TCT CTAGGA GAA AAT GTG GTT 96 Ser Leu Val Pro Gln Arg His Gly Pro Ser Leu GlyGlu Asn Val Val 20 25 30 TAT CGT TCT GAA AGC CCC AAC TCA CAG GCG GAT GTAGGA AGA CCT CTG 144 Tyr Arg Ser Glu Ser Pro Asn Ser Gln Ala Asp Val GlyArg Pro Leu 35 40 45 TCT GGA TCC GGC ATT GCA GCA TTT GCT CAA GCT CAC CCAAGC AAT GGA 192 Ser Gly Ser Gly Ile Ala Ala Phe Ala Gln Ala His Pro SerAsn Gly 50 55 60 CAG AGA GTG AAC CCC CCA CCA CCA CCT CAA GTT AGG AGT GTTACT CCT 240 Gln Arg Val Asn Pro Pro Pro Pro Pro Gln Val Arg Ser Val ThrPro 65 70 75 80 CCA CCA CCT CCG AGA GGC CAG ACC CCA CCT CCC CGA GGC ACCACT CCC 288 Pro Pro Pro Pro Arg Gly Gln Thr Pro Pro Pro Arg Gly Thr ThrPro 85 90 95 CCT CCC CCC TCA TGG GAA CCA AGC TCT CAG ACA AAG CGC TAC TCTGGG 336 Pro Pro Pro Ser Trp Glu Pro Ser Ser Gln Thr Lys Arg Tyr Ser Gly100 105 110 AAC ATG GAG TAC GTA ATC TCC CGA ATC TCC CCT GTT CCA CCT GGGGCG 384 Asn Met Glu Tyr Val Ile Ser Arg Ile Ser Pro Val Pro Pro Gly Ala115 120 125 TGG CAG GAG GGG TAC CCT CCA CCA CCT CTT ACC ACT TCT CCC ATGAAT 432 Trp Gln Glu Gly Tyr Pro Pro Pro Pro Leu Thr Thr Ser Pro Met Asn130 135 140 CCC CCT AGC CAG GCT CAG AGG GCC ATT AGT TCT GTT CCA GTT GGTAGA 480 Pro Pro Ser Gln Ala Gln Arg Ala Ile Ser Ser Val Pro Val Gly Arg145 150 155 160 CAA CCC ATC ATC ATG CAG AGT ACT AGC AAA TTT AAC TTT ACACCA GGG 528 Gln Pro Ile Ile Met Gln Ser Thr Ser Lys Phe Asn Phe Thr ProGly 165 170 175 CGA CCT GGA GTT CAG AAT GGT GGT GGT CAG TCT GAT TTT ATCGTG CAC 576 Arg Pro Gly Val Gln Asn Gly Gly Gly Gln Ser Asp Phe Ile ValHis 180 185 190 CAA AAT GTC CCC ACT GGT TCT GTG ACT CGG CAG CCA CCA CCTCCA TAT 624 Gln Asn Val Pro Thr Gly Ser Val Thr Arg Gln Pro Pro Pro ProTyr 195 200 205 CCT CTG ACC CCA GCT AAT GGA CAA AGC CCC TCT GCT TTA CAAACA GGG 672 Pro Leu Thr Pro Ala Asn Gly Gln Ser Pro Ser Ala Leu Gln ThrGly 210 215 220 GCT TCT GCT GCT CCA CCA TCA TTC GCC AAT GGA AAC GTT CCTCAG TCG 720 Ala Ser Ala Ala Pro Pro Ser Phe Ala Asn Gly Asn Val Pro GlnSer 225 230 235 240 ATG ATG GTG CCC AAC AGG AAC AGT CAT AAC ATG GAG CTTTAT AAT ATT 768 Met Met Val Pro Asn Arg Asn Ser His Asn Met Glu Leu TyrAsn Ile 245 250 255 AAT GTC CCT GGA CTG CAA ACA GCC TGG CCC CAG TCG TCTTCT GCT CCT 816 Asn Val Pro Gly Leu Gln Thr Ala Trp Pro Gln Ser Ser SerAla Pro 260 265 270 GCG CAG TCA TCC CCA AGC GGT GGG CAT GAA ATT CCT ACATGG CAA CCT 864 Ala Gln Ser Ser Pro Ser Gly Gly His Glu Ile Pro Thr TrpGln Pro 275 280 285 AAC ATA CCA GTG AGG TCA AAT TCT TTT AAT AAC CCA TTAGGA AGT AGA 912 Asn Ile Pro Val Arg Ser Asn Ser Phe Asn Asn Pro Leu GlySer Arg 290 295 300 GCA AGT CAC TCT GCT AAT TCT CAG CCT TCT GCC ACT ACAGTC ACT GCC 960 Ala Ser His Ser Ala Asn Ser Gln Pro Ser Ala Thr Thr ValThr Ala 305 310 315 320 ATC ACA CCC GCT CCT ATT CAA CAG CCC GTG AAA AGCATG CGC GTC CTG 1008 Ile Thr Pro Ala Pro Ile Gln Gln Pro Val Lys Ser MetArg Val Leu 325 330 335 AAA CCA GAG CTG CAG ACT GCT TTA GCC CCA ACC CATCCT TCT TGG ATG 1056 Lys Pro Glu Leu Gln Thr Ala Leu Ala Pro Thr His ProSer Trp Met 340 345 350 CCA CAG CCA GTT CAG ACT GTT CAG CCT ACC CCT TTTTCT GAG GGT ACA 1104 Pro Gln Pro Val Gln Thr Val Gln Pro Thr Pro Phe SerGlu Gly Thr 355 360 365 GCT TCA AGT GTG CCT GTC ATC CCA CCT GTT GCT GAAGCT CCA AGC TAT 1152 Ala Ser Ser Val Pro Val Ile Pro Pro Val Ala Glu AlaPro Ser Tyr 370 375 380 CAA GGT CCA CCA CCG CCT TAT CCA AAA CAT CTG CTACAC CAA AAC CCA 1200 Gln Gly Pro Pro Pro Pro Tyr Pro Lys His Leu Leu HisGln Asn Pro 385 390 395 400 TCT GTC CCT CCA TAT GAG TCA GTA AGT AAG CCCTGC AAA GAT GAA CAG 1248 Ser Val Pro Pro Tyr Glu Ser Val Ser Lys Pro CysLys Asp Glu Gln 405 410 415 CCT AGC TTA CCC AAG GAA GAT GAT AGT GAG AAGAGT GCG GAC AGT GGT 1296 Pro Ser Leu Pro Lys Glu Asp Asp Ser Glu Lys SerAla Asp Ser Gly 420 425 430 GAC TCT GGG GAT AAA GAA AAG AAA CAG ATT ACAACT TCA CCT ATC ACT 1344 Asp Ser Gly Asp Lys Glu Lys Lys Gln Ile Thr ThrSer Pro Ile Thr 435 440 445 GTT CGG AAA AAC AAG AAA GAT GAA GAA CGA AGAGAG TCT CGG ATT CAG 1392 Val Arg Lys Asn Lys Lys Asp Glu Glu Arg Arg GluSer Arg Ile Gln 450 455 460 AGT TAC TCC CCA CAG GCC TTT AAG TTC TTC ATGGAG CAG CAC GTA GAG 1440 Ser Tyr Ser Pro Gln Ala Phe Lys Phe Phe Met GluGln His Val Glu 465 470 475 480 AAC GTC CTG AAG TCT CAT CAG CAG CGT CTGCAT CGG AAG AAG CAG CTA 1488 Asn Val Leu Lys Ser His Gln Gln Arg Leu HisArg Lys Lys Gln Leu 485 490 495 GAA AAT GAA ATG ATG CGG GTT GGA TTA TCTCAA GAT GCC CAG GAT CAA 1536 Glu Asn Glu Met Met Arg Val Gly Leu Ser GlnAsp Ala Gln Asp Gln 500 505 510 ATG AGA AAG ATG CTT TGC CAG AAA GAG TCTAAC TAT ATT CGT CTT AAA 1584 Met Arg Lys Met Leu Cys Gln Lys Glu Ser AsnTyr Ile Arg Leu Lys 515 520 525 AGG GCT AAA ATG GAC AAG TCT ATG TTT GTAAAG ATA AAG ACA TTA GGA 1632 Arg Ala Lys Met Asp Lys Ser Met Phe Val LysIle Lys Thr Leu Gly 530 535 540 ATA GGA GCG TTT GGT GAA GTC TGT CTA GCAAGA AAA GTC GAT ACT AAA 1680 Ile Gly Ala Phe Gly Glu Val Cys Leu Ala ArgLys Val Asp Thr Lys 545 550 555 560 GCT TTG TAT GCA ACA AAG ACT CTT CGAAAG AAA GAC GTT CTG CTC CGA 1728 Ala Leu Tyr Ala Thr Lys Thr Leu Arg LysLys Asp Val Leu Leu Arg 565 570 575 AAT CAG GTG GCT CAT GTG AAA GCG GAGAGG GAT ATC CTA GCA GAA GCC 1776 Asn Gln Val Ala His Val Lys Ala Glu ArgAsp Ile Leu Ala Glu Ala 580 585 590 GAC AAT GAG TGG GTG GTC CGC CTG TACTAC TCT TTC CAG GAC AAG GAC 1824 Asp Asn Glu Trp Val Val Arg Leu Tyr TyrSer Phe Gln Asp Lys Asp 595 600 605 AAC TTG TAC TTT GTG ATG GAC TAC ATTCCT GGG GGG GAT ATG ATG AGC 1872 Asn Leu Tyr Phe Val Met Asp Tyr Ile ProGly Gly Asp Met Met Ser 610 615 620 CTA TTA ATT AGA ATG GGC ATC TTT CCTGAA AAT CTG GCA CGA TTC TAC 1920 Leu Leu Ile Arg Met Gly Ile Phe Pro GluAsn Leu Ala Arg Phe Tyr 625 630 635 640 ATA GCA GAA CTT ACC TGT GCA GTTGAA AGT GTT CAT AAA ATG GGT TTT 1968 Ile Ala Glu Leu Thr Cys Ala Val GluSer Val His Lys Met Gly Phe 645 650 655 ATT CAT AGA GAT ATT AAA CCT GATAAC ATT TTG ATT GAC CGT GAT GGC 2016 Ile His Arg Asp Ile Lys Pro Asp AsnIle Leu Ile Asp Arg Asp Gly 660 665 670 CAT ATT AAA TTG ACT GAC TTT GGCTTG TGC ACT GGC TTC AGA TGG ACA 2064 His Ile Lys Leu Thr Asp Phe Gly LeuCys Thr Gly Phe Arg Trp Thr 675 680 685 CAT GAC TCC AAG TAC TAC CAG AGTGGG GAT CAC CCA CGG CAA GAT AGC 2112 His Asp Ser Lys Tyr Tyr Gln Ser GlyAsp His Pro Arg Gln Asp Ser 690 695 700 ATG GAT TTC AGT AAC GAA TGG GGAGAT CCT TCC AAT TGT CGG TGT GGG 2160 Met Asp Phe Ser Asn Glu Trp Gly AspPro Ser Asn Cys Arg Cys Gly 705 710 715 720 GAC AGA CTG AAG CCA CTG GAGCGG AGA GCT GCT CGC CAG CAC CAG CGA 2208 Asp Arg Leu Lys Pro Leu Glu ArgArg Ala Ala Arg Gln His Gln Arg 725 730 735 TGT CTA GCC CAT TCT CTG GTTGGG ACT CCC AAT TAT ATT GCA CCT GAA 2256 Cys Leu Ala His Ser Leu Val GlyThr Pro Asn Tyr Ile Ala Pro Glu 740 745 750 GTG CTA CTG CGA ACA GGA TATACA CAG CTG TGT GAC TGG TGG AGT GTT 2304 Val Leu Leu Arg Thr Gly Tyr ThrGln Leu Cys Asp Trp Trp Ser Val 755 760 765 GGT GTT ATT CTT TGT GAA ATGTTG GTG GGA CAA CCT CCT TTC TTG GCA 2352 Gly Val Ile Leu Cys Glu Met LeuVal Gly Gln Pro Pro Phe Leu Ala 770 775 780 CAA ACC CCA TTA GAA ACA CAAATG AAG GTT ATC ATC TGG CAA ACT TCT 2400 Gln Thr Pro Leu Glu Thr Gln MetLys Val Ile Ile Trp Gln Thr Ser 785 790 795 800 CTA CAC ATC CCT CCT CAAGCT AAG CTG AGT CCT GAA GCC TCT GAC CTC 2448 Leu His Ile Pro Pro Gln AlaLys Leu Ser Pro Glu Ala Ser Asp Leu 805 810 815 ATT ATC AAA CTG TGT CGAGGA CCA GAA GAC CGC CTC GGC AAG AAC GGT 2496 Ile Ile Lys Leu Cys Arg GlyPro Glu Asp Arg Leu Gly Lys Asn Gly 820 825 830 GCT GAT GAG ATA AAG GCTCAT CCA TTT TTT AAG ACC ATC GAT TTC TCT 2544 Ala Asp Glu Ile Lys Ala HisPro Phe Phe Lys Thr Ile Asp Phe Ser 835 840 845 AGT GAT CTG AGA CAG CAGTCT GCT TCA TAC ATC CCT AAA ATC ACG CAT 2592 Ser Asp Leu Arg Gln Gln SerAla Ser Tyr Ile Pro Lys Ile Thr His 850 855 860 CCA ACA GAT ACA TCC AATTTC GAC CCT GTT GAT CCT GAT AAA TTG TGG 2640 Pro Thr Asp Thr Ser Asn PheAsp Pro Val Asp Pro Asp Lys Leu Trp 865 870 875 880 AGC GAT GGC AGC GAGGAG GAA AAT ATC AGT GAC ACT CTG AGC GGA TGG 2688 Ser Asp Gly Ser Glu GluGlu Asn Ile Ser Asp Thr Leu Ser Gly Trp 885 890 895 TAT AAA AAT GGG AAGCAC CCC GAG CAC GCT TTC TAT GAG TTC ACC TTT 2736 Tyr Lys Asn Gly Lys HisPro Glu His Ala Phe Tyr Glu Phe Thr Phe 900 905 910 CGG AGG TTT TTT GATGAC AAT GGC TAC CCA TAT AAT TAT CCA AAG CCT 2784 Arg Arg Phe Phe Asp AspAsn Gly Tyr Pro Tyr Asn Tyr Pro Lys Pro 915 920 925 ATT GAG TAT GAA TACATT CAT TCA CAG GGC TCA GAA CAA CAG TCT GAT 2832 Ile Glu Tyr Glu Tyr IleHis Ser Gln Gly Ser Glu Gln Gln Ser Asp 930 935 940 GAA GAT GAT CAA CACACA AGC TCC GAT GGA AAC AAC CGA GAT CTA GTG 2880 Glu Asp Asp Gln His ThrSer Ser Asp Gly Asn Asn Arg Asp Leu Val 945 950 955 960 TAT GTT TAATAAACTAGGA GATCATTGTA AGAATTTGCA AGAGGCCTGA 2929 Tyr Val AGTGCAGGGGTTTTTGAAGT TTTGAGAAAA TTATGCAAAT GTGACAGAGT TTGTGTGCTC 2989 TGTGTACAATATTTTATTTT CCTAAGTTAT GGGAAATTGT TTTAAAATGT TAATTTATTC 3049 CACCCTTTTAATTCAGTAAT TTAGAAAAAA TTGTTATAAG GAAAGTAAAT TATGAACTGA 3109 GTATTATAGTCAATTCTTGG TACTTAAAGT ACTTAAAAAG AGAAGCCTGG TATCTTTTGT 3169 ATATATAATAAATAATTTTA AAATCCCAAA AAAAAAAAAA AAAA 3213 962 amino acids amino acidunknown protein 6 Val Gln His Ser Ile Asn Arg Lys Gln Ser Trp Lys GlySer Lys Glu 1 5 10 15 Ser Leu Val Pro Gln Arg His Gly Pro Ser Leu GlyGlu Asn Val Val 20 25 30 Tyr Arg Ser Glu Ser Pro Asn Ser Gln Ala Asp ValGly Arg Pro Leu 35 40 45 Ser Gly Ser Gly Ile Ala Ala Phe Ala Gln Ala HisPro Ser Asn Gly 50 55 60 Gln Arg Val Asn Pro Pro Pro Pro Pro Gln Val ArgSer Val Thr Pro 65 70 75 80 Pro Pro Pro Pro Arg Gly Gln Thr Pro Pro ProArg Gly Thr Thr Pro 85 90 95 Pro Pro Pro Ser Trp Glu Pro Ser Ser Gln ThrLys Arg Tyr Ser Gly 100 105 110 Asn Met Glu Tyr Val Ile Ser Arg Ile SerPro Val Pro Pro Gly Ala 115 120 125 Trp Gln Glu Gly Tyr Pro Pro Pro ProLeu Thr Thr Ser Pro Met Asn 130 135 140 Pro Pro Ser Gln Ala Gln Arg AlaIle Ser Ser Val Pro Val Gly Arg 145 150 155 160 Gln Pro Ile Ile Met GlnSer Thr Ser Lys Phe Asn Phe Thr Pro Gly 165 170 175 Arg Pro Gly Val GlnAsn Gly Gly Gly Gln Ser Asp Phe Ile Val His 180 185 190 Gln Asn Val ProThr Gly Ser Val Thr Arg Gln Pro Pro Pro Pro Tyr 195 200 205 Pro Leu ThrPro Ala Asn Gly Gln Ser Pro Ser Ala Leu Gln Thr Gly 210 215 220 Ala SerAla Ala Pro Pro Ser Phe Ala Asn Gly Asn Val Pro Gln Ser 225 230 235 240Met Met Val Pro Asn Arg Asn Ser His Asn Met Glu Leu Tyr Asn Ile 245 250255 Asn Val Pro Gly Leu Gln Thr Ala Trp Pro Gln Ser Ser Ser Ala Pro 260265 270 Ala Gln Ser Ser Pro Ser Gly Gly His Glu Ile Pro Thr Trp Gln Pro275 280 285 Asn Ile Pro Val Arg Ser Asn Ser Phe Asn Asn Pro Leu Gly SerArg 290 295 300 Ala Ser His Ser Ala Asn Ser Gln Pro Ser Ala Thr Thr ValThr Ala 305 310 315 320 Ile Thr Pro Ala Pro Ile Gln Gln Pro Val Lys SerMet Arg Val Leu 325 330 335 Lys Pro Glu Leu Gln Thr Ala Leu Ala Pro ThrHis Pro Ser Trp Met 340 345 350 Pro Gln Pro Val Gln Thr Val Gln Pro ThrPro Phe Ser Glu Gly Thr 355 360 365 Ala Ser Ser Val Pro Val Ile Pro ProVal Ala Glu Ala Pro Ser Tyr 370 375 380 Gln Gly Pro Pro Pro Pro Tyr ProLys His Leu Leu His Gln Asn Pro 385 390 395 400 Ser Val Pro Pro Tyr GluSer Val Ser Lys Pro Cys Lys Asp Glu Gln 405 410 415 Pro Ser Leu Pro LysGlu Asp Asp Ser Glu Lys Ser Ala Asp Ser Gly 420 425 430 Asp Ser Gly AspLys Glu Lys Lys Gln Ile Thr Thr Ser Pro Ile Thr 435 440 445 Val Arg LysAsn Lys Lys Asp Glu Glu Arg Arg Glu Ser Arg Ile Gln 450 455 460 Ser TyrSer Pro Gln Ala Phe Lys Phe Phe Met Glu Gln His Val Glu 465 470 475 480Asn Val Leu Lys Ser His Gln Gln Arg Leu His Arg Lys Lys Gln Leu 485 490495 Glu Asn Glu Met Met Arg Val Gly Leu Ser Gln Asp Ala Gln Asp Gln 500505 510 Met Arg Lys Met Leu Cys Gln Lys Glu Ser Asn Tyr Ile Arg Leu Lys515 520 525 Arg Ala Lys Met Asp Lys Ser Met Phe Val Lys Ile Lys Thr LeuGly 530 535 540 Ile Gly Ala Phe Gly Glu Val Cys Leu Ala Arg Lys Val AspThr Lys 545 550 555 560 Ala Leu Tyr Ala Thr Lys Thr Leu Arg Lys Lys AspVal Leu Leu Arg 565 570 575 Asn Gln Val Ala His Val Lys Ala Glu Arg AspIle Leu Ala Glu Ala 580 585 590 Asp Asn Glu Trp Val Val Arg Leu Tyr TyrSer Phe Gln Asp Lys Asp 595 600 605 Asn Leu Tyr Phe Val Met Asp Tyr IlePro Gly Gly Asp Met Met Ser 610 615 620 Leu Leu Ile Arg Met Gly Ile PhePro Glu Asn Leu Ala Arg Phe Tyr 625 630 635 640 Ile Ala Glu Leu Thr CysAla Val Glu Ser Val His Lys Met Gly Phe 645 650 655 Ile His Arg Asp IleLys Pro Asp Asn Ile Leu Ile Asp Arg Asp Gly 660 665 670 His Ile Lys LeuThr Asp Phe Gly Leu Cys Thr Gly Phe Arg Trp Thr 675 680 685 His Asp SerLys Tyr Tyr Gln Ser Gly Asp His Pro Arg Gln Asp Ser 690 695 700 Met AspPhe Ser Asn Glu Trp Gly Asp Pro Ser Asn Cys Arg Cys Gly 705 710 715 720Asp Arg Leu Lys Pro Leu Glu Arg Arg Ala Ala Arg Gln His Gln Arg 725 730735 Cys Leu Ala His Ser Leu Val Gly Thr Pro Asn Tyr Ile Ala Pro Glu 740745 750 Val Leu Leu Arg Thr Gly Tyr Thr Gln Leu Cys Asp Trp Trp Ser Val755 760 765 Gly Val Ile Leu Cys Glu Met Leu Val Gly Gln Pro Pro Phe LeuAla 770 775 780 Gln Thr Pro Leu Glu Thr Gln Met Lys Val Ile Ile Trp GlnThr Ser 785 790 795 800 Leu His Ile Pro Pro Gln Ala Lys Leu Ser Pro GluAla Ser Asp Leu 805 810 815 Ile Ile Lys Leu Cys Arg Gly Pro Glu Asp ArgLeu Gly Lys Asn Gly 820 825 830 Ala Asp Glu Ile Lys Ala His Pro Phe PheLys Thr Ile Asp Phe Ser 835 840 845 Ser Asp Leu Arg Gln Gln Ser Ala SerTyr Ile Pro Lys Ile Thr His 850 855 860 Pro Thr Asp Thr Ser Asn Phe AspPro Val Asp Pro Asp Lys Leu Trp 865 870 875 880 Ser Asp Gly Ser Glu GluGlu Asn Ile Ser Asp Thr Leu Ser Gly Trp 885 890 895 Tyr Lys Asn Gly LysHis Pro Glu His Ala Phe Tyr Glu Phe Thr Phe 900 905 910 Arg Arg Phe PheAsp Asp Asn Gly Tyr Pro Tyr Asn Tyr Pro Lys Pro 915 920 925 Ile Glu TyrGlu Tyr Ile His Ser Gln Gly Ser Glu Gln Gln Ser Asp 930 935 940 Glu AspAsp Gln His Thr Ser Ser Asp Gly Asn Asn Arg Asp Leu Val 945 950 955 960Tyr Val 3154 amino acids nucleic acid double unknown cDNA CDS 1..2943 7ATG AGA GCC ACC CCG AAG TTT GGA CCT TAT CAA AAA GCT CTC AGG GAA 48 MetArg Ala Thr Pro Lys Phe Gly Pro Tyr Gln Lys Ala Leu Arg Glu 1 5 10 15ATC CGA TAT TCC CTC CTG CCT TTT GCC AAC GAG TCA GGC ACT TCG GCA 96 IleArg Tyr Ser Leu Leu Pro Phe Ala Asn Glu Ser Gly Thr Ser Ala 20 25 30 GCTGCA GAG GTG AAC CGG CAG ATG CTT CAG GAG TTG GTG AAT GCG GCA 144 Ala AlaGlu Val Asn Arg Gln Met Leu Gln Glu Leu Val Asn Ala Ala 35 40 45 TGT GACCAG GAG ATG GCT GGC AGA GCG CTC ACG CAG ACG GGC AGT AGG 192 Cys Asp GlnGlu Met Ala Gly Arg Ala Leu Thr Gln Thr Gly Ser Arg 50 55 60 AGT ATC GAAGCT GCC TTG GAG TAC ATC AGT AAG ATG GGC TAC CTG GAC 240 Ser Ile Glu AlaAla Leu Glu Tyr Ile Ser Lys Met Gly Tyr Leu Asp 65 70 75 80 CCC AGG AATGAG CAG ATT GTG CGA GTC ATC AAG CAG ACC TCC CCA GGA 288 Pro Arg Asn GluGln Ile Val Arg Val Ile Lys Gln Thr Ser Pro Gly 85 90 95 AAG GGC CTG GCGTCC ACC CCG GTG ACT CGG CGG CCC AGT TTC GAG GGC 336 Lys Gly Leu Ala SerThr Pro Val Thr Arg Arg Pro Ser Phe Glu Gly 100 105 110 ACA GGG GAA GCACTC CCA TCC TAC CAC CAG CTG GGT GGT GCA AAC TAC 384 Thr Gly Glu Ala LeuPro Ser Tyr His Gln Leu Gly Gly Ala Asn Tyr 115 120 125 GAG GGC CCC GCCGCA CTG GAG GAG ATG CCG CGG CAA TAT TTA GAC TTT 432 Glu Gly Pro Ala AlaLeu Glu Glu Met Pro Arg Gln Tyr Leu Asp Phe 130 135 140 CTC TTC CCT GGAGCC GGA GCC GGC ACC CAC GGT GCC CAG GCT CAC CAG 480 Leu Phe Pro Gly AlaGly Ala Gly Thr His Gly Ala Gln Ala His Gln 145 150 155 160 CAT CCT CCCAAA GGG TAC AGC ACA GCA GTA GAG CCA AGT GCG CAC TTT 528 His Pro Pro LysGly Tyr Ser Thr Ala Val Glu Pro Ser Ala His Phe 165 170 175 CCG GGC ACACAC TAT GGT CGT GGT CAT CTA CTA TCG GAG CAG TCT GGG 576 Pro Gly Thr HisTyr Gly Arg Gly His Leu Leu Ser Glu Gln Ser Gly 180 185 190 TAT GGG GTGCAG CGC AGT TCC TCC TTC CAG AAC AAG ACG CCA CCA GAT 624 Tyr Gly Val GlnArg Ser Ser Ser Phe Gln Asn Lys Thr Pro Pro Asp 195 200 205 GCC TAT TCCAGC ATG GCC AAG GCC CAG GGT GGC CCT CCC GCC AGC CTC 672 Ala Tyr Ser SerMet Ala Lys Ala Gln Gly Gly Pro Pro Ala Ser Leu 210 215 220 ACC TTT CCTGCC CAT GCT GGG CTG TAC ACT GCC TCG CAC CAC AAG CCG 720 Thr Phe Pro AlaHis Ala Gly Leu Tyr Thr Ala Ser His His Lys Pro 225 230 235 240 GCG GCTACC CCA CCT GGG GCC CAC CCA TTA CAT GTG TTG GGC ACC CGG 768 Ala Ala ThrPro Pro Gly Ala His Pro Leu His Val Leu Gly Thr Arg 245 250 255 GGT CCCACG TTT ACT GGC GAA AGC TCT GCA CAG GCT GTG CTG GCA CCG 816 Gly Pro ThrPhe Thr Gly Glu Ser Ser Ala Gln Ala Val Leu Ala Pro 260 265 270 TCC AGGAAC AGC CTC AAT GCT GAC TTG TAC GAG CTG GGC TCC ACG GTG 864 Ser Arg AsnSer Leu Asn Ala Asp Leu Tyr Glu Leu Gly Ser Thr Val 275 280 285 CCC TGGTCT GCA GCT CCA CTG GCA CGC CGC GAC TCG CTG CAG AAG CAG 912 Pro Trp SerAla Ala Pro Leu Ala Arg Arg Asp Ser Leu Gln Lys Gln 290 295 300 GGT CTAGAA GCC TCG CGG CCG CAT GTG GCT TTT CGG GCT GGC CCC AGC 960 Gly Leu GluAla Ser Arg Pro His Val Ala Phe Arg Ala Gly Pro Ser 305 310 315 320 AGGACC AAC TCC TTC AAC AAC CCA CAA CCT GAG CCC TCA CTG CCC GCC 1008 Arg ThrAsn Ser Phe Asn Asn Pro Gln Pro Glu Pro Ser Leu Pro Ala 325 330 335 CCCAAC ACG GTC ACC GCC GTG ACG GCC GCA CAC ATC CTT CAC CCT GTG 1056 Pro AsnThr Val Thr Ala Val Thr Ala Ala His Ile Leu His Pro Val 340 345 350 AAGAGC GTG CGT GTG CTG CGG CCC GAG CCC CAG ACA GCC GTG GGG CCC 1104 Lys SerVal Arg Val Leu Arg Pro Glu Pro Gln Thr Ala Val Gly Pro 355 360 365 TCGCAC CCC GCC TGG GTG GCT GCG CCC ACA GCA CCT GCC ACT GAG AGC 1152 Ser HisPro Ala Trp Val Ala Ala Pro Thr Ala Pro Ala Thr Glu Ser 370 375 380 CTGGAG ACG AAG GAG GGC AGC GCA GGC CCA CAC CCG CTG GAT GTG GAC 1200 Leu GluThr Lys Glu Gly Ser Ala Gly Pro His Pro Leu Asp Val Asp 385 390 395 400TAT GGC GGC TCC GAG CGC AGG TGC CCA CCG CCT CCG TAT CCA AAG CAC 1248 TyrGly Gly Ser Glu Arg Arg Cys Pro Pro Pro Pro Tyr Pro Lys His 405 410 415TTG CTG CTG CCC AGT AAG TCT GAG CAG TAC AGC GTG GAC CTG GAC AGC 1296 LeuLeu Leu Pro Ser Lys Ser Glu Gln Tyr Ser Val Asp Leu Asp Ser 420 425 430CTG TGC ACC AGT GTG CAG CAG AGT CTG CGA GGG GGC ACT GAT CTA GAC 1344 LeuCys Thr Ser Val Gln Gln Ser Leu Arg Gly Gly Thr Asp Leu Asp 435 440 445GGG AGT GAC AAG AGC CAC AAA GGT GCG AAG GGA GAC AAA GCT GGC AGA 1392 GlySer Asp Lys Ser His Lys Gly Ala Lys Gly Asp Lys Ala Gly Arg 450 455 460GAC AAA AAG CAG ATT CAG ACC TCC CCG GTG CCT GTC CGC AAG AAT AGC 1440 AspLys Lys Gln Ile Gln Thr Ser Pro Val Pro Val Arg Lys Asn Ser 465 470 475480 AGA GAT GAA GAG AAG AGA GAG TCT CGC ATC AAG AGT TAC TCC CCT TAT 1488Arg Asp Glu Glu Lys Arg Glu Ser Arg Ile Lys Ser Tyr Ser Pro Tyr 485 490495 GCC TTC AAA TTC TTC ATG GAG CAA CAC GTG GAG AAT GTC ATC AAA ACC 1536Ala Phe Lys Phe Phe Met Glu Gln His Val Glu Asn Val Ile Lys Thr 500 505510 TAC CAG CAG AAG GTC AGC CGG AGG CTA CAG CTG GAG CAG GAA ATG GCC 1584Tyr Gln Gln Lys Val Ser Arg Arg Leu Gln Leu Glu Gln Glu Met Ala 515 520525 AAA GCT GGG CTC TGT GAG GCC GAG CAG GAG CAG ATG AGG AAG ATC CTC 1632Lys Ala Gly Leu Cys Glu Ala Glu Gln Glu Gln Met Arg Lys Ile Leu 530 535540 TAC CAG AAG GAG TCT AAC TAC AAC CGG CTG AAG AGG GCC AAG ATG GAC 1680Tyr Gln Lys Glu Ser Asn Tyr Asn Arg Leu Lys Arg Ala Lys Met Asp 545 550555 560 AAG TCC ATG TTT GTG AAA ATC AAG ACT CTA GGC ATC GGT GCC TTT GGG1728 Lys Ser Met Phe Val Lys Ile Lys Thr Leu Gly Ile Gly Ala Phe Gly 565570 575 GAA GTG TGC CTC GCT TGT AAG CTG GAC ACT CAC GCT CTG TAC GCC ATG1776 Glu Val Cys Leu Ala Cys Lys Leu Asp Thr His Ala Leu Tyr Ala Met 580585 590 AAG ACT CTC AGG AAG AAG GAT GTC CTG AAC CGG AAT CAA GTG GCC CAT1824 Lys Thr Leu Arg Lys Lys Asp Val Leu Asn Arg Asn Gln Val Ala His 595600 605 GTC AAG GCT GAG AGG GAC ATC CTG GCT GAA GCA GAC AAT GAG TGG GTG1872 Val Lys Ala Glu Arg Asp Ile Leu Ala Glu Ala Asp Asn Glu Trp Val 610615 620 GTC AAA CTC TAC TAC TCC TTC CAG GAC AAG GAC AGC CTG TAC TTT GTG1920 Val Lys Leu Tyr Tyr Ser Phe Gln Asp Lys Asp Ser Leu Tyr Phe Val 625630 635 640 ATG GAC TAC ATA CCA GGC GGG GAT ATG ATG AGC CTG CTG ATC AGGATG 1968 Met Asp Tyr Ile Pro Gly Gly Asp Met Met Ser Leu Leu Ile Arg Met645 650 655 GAG GTC TTC CCT GAG CAC CTG GCC CGC TTC TAC ATT GCA GAG TTGACC 2016 Glu Val Phe Pro Glu His Leu Ala Arg Phe Tyr Ile Ala Glu Leu Thr660 665 670 CTG GCC ATT GAA AGT GTC CAC AAG ATG GGC TTT ATC CAC CGG GACATC 2064 Leu Ala Ile Glu Ser Val His Lys Met Gly Phe Ile His Arg Asp Ile675 680 685 AAG CCT GAC AAC ATA CTC ATC GAC CTG GAT GGT CAT ATT AAG CTGACA 2112 Lys Pro Asp Asn Ile Leu Ile Asp Leu Asp Gly His Ile Lys Leu Thr690 695 700 GAT TTT GGC CTC TGC ACT GGA TTC AGG TGG ACT CAC AAT TCC AAGTAC 2160 Asp Phe Gly Leu Cys Thr Gly Phe Arg Trp Thr His Asn Ser Lys Tyr705 710 715 720 TAC CAG AAA GGG AAC CAC ATG AGA CAG GAC AGC ATG GAG CCCGGT GAC 2208 Tyr Gln Lys Gly Asn His Met Arg Gln Asp Ser Met Glu Pro GlyAsp 725 730 735 CTC TGG GAC GAT GTT TCC AAC TGT CGC TGT GGA GAC AGG TTAAAG ACC 2256 Leu Trp Asp Asp Val Ser Asn Cys Arg Cys Gly Asp Arg Leu LysThr 740 745 750 CTG GAG CAG AGG GCG CAG AAG CAG CAC CAG AGG TGC CTG GCACAT TCT 2304 Leu Glu Gln Arg Ala Gln Lys Gln His Gln Arg Cys Leu Ala HisSer 755 760 765 CTT GTC GGG ACA CCA AAT TAC ATC GCT CCG GAG GTG CTT CTCCGC AAA 2352 Leu Val Gly Thr Pro Asn Tyr Ile Ala Pro Glu Val Leu Leu ArgLys 770 775 780 GGG TAC ACG CAG CTC TGT GAC TGG TGG AGC GTC GGT GTG ATTCTC TTT 2400 Gly Tyr Thr Gln Leu Cys Asp Trp Trp Ser Val Gly Val Ile LeuPhe 785 790 795 800 GAG ATG CTG GTT GGG CAG CCG CCT TTC TTG GCC CCC ACCCCC ACA GAG 2448 Glu Met Leu Val Gly Gln Pro Pro Phe Leu Ala Pro Thr ProThr Glu 805 810 815 ACG CAG CTG AAG GTG ATC AAC TGG GAG AGC ACG CTG CATATC CCT ACG 2496 Thr Gln Leu Lys Val Ile Asn Trp Glu Ser Thr Leu His IlePro Thr 820 825 830 CAG GTG AGG CTC AGC GCT GAG GCC CGA GAC CTC ATC ACGAAG CTG TGC 2544 Gln Val Arg Leu Ser Ala Glu Ala Arg Asp Leu Ile Thr LysLeu Cys 835 840 845 TGC GCG GCT GAC TGC CGC CTG GGC AGG GAT GGG GCA GATGAC CTC AAG 2592 Cys Ala Ala Asp Cys Arg Leu Gly Arg Asp Gly Ala Asp AspLeu Lys 850 855 860 GCA CAC CCG TTC TTC AAC ACC ATC GAC TTT TCC CGT GACATC CGA AAG 2640 Ala His Pro Phe Phe Asn Thr Ile Asp Phe Ser Arg Asp IleArg Lys 865 870 875 880 CAG GCT GCA CCC TAC GTC CCC ACC ATC AGC CAC CCCATG GAC ACC TCC 2688 Gln Ala Ala Pro Tyr Val Pro Thr Ile Ser His Pro MetAsp Thr Ser 885 890 895 AAT TTT GAC CCG GTG GAT GAA GAA AGC CCC TGG CACGAG GCC AGC GGA 2736 Asn Phe Asp Pro Val Asp Glu Glu Ser Pro Trp His GluAla Ser Gly 900 905 910 GAG AGC GCC AAG GCC TGG GAC ACG CTG GCC TCC CCCAGC AGC AAG CAT 2784 Glu Ser Ala Lys Ala Trp Asp Thr Leu Ala Ser Pro SerSer Lys His 915 920 925 CCA GAG CAC GCC TTC TAT GAG TTC ACC TTC CGC AGGTTC TTC GAT GAC 2832 Pro Glu His Ala Phe Tyr Glu Phe Thr Phe Arg Arg PhePhe Asp Asp 930 935 940 AAC GGC TAT CCC TTC CGG TGC CCG AAG CCC TCA GAGCCC GCA GAG AGT 2880 Asn Gly Tyr Pro Phe Arg Cys Pro Lys Pro Ser Glu ProAla Glu Ser 945 950 955 960 GCA GAC CCA GGG GAT GCG GAC TTG GAA GGT GCGGCC GAG GGC TGC CAG 2928 Ala Asp Pro Gly Asp Ala Asp Leu Glu Gly Ala AlaGlu Gly Cys Gln 965 970 975 CCG GTG TAC GTG TAA GCCTCAGTTA ACCACAACTCGAGGAAACCC AAAATGAGAT 2983 Pro Val Tyr Val 980 TTCTTTTCAG AAGACAAACTCAAGCTTAGG AATCCTTCAT TTTTAGTTCT GGTAAATGGG 3043 CAACAGGAAG AGTCAACATGATTTCAAATT AGCCCTCTGA GGACCTTCAC TGCATTAAAA 3103 CAGTATTTTT TAAAAAATTAGTACAGTATG GAAAGAGCAC TTATTTTGGG GG 3155 980 amino acids amino acidunknown protein 8 Met Arg Ala Thr Pro Lys Phe Gly Pro Tyr Gln Lys AlaLeu Arg Glu 1 5 10 15 Ile Arg Tyr Ser Leu Leu Pro Phe Ala Asn Glu SerGly Thr Ser Ala 20 25 30 Ala Ala Glu Val Asn Arg Gln Met Leu Gln Glu LeuVal Asn Ala Ala 35 40 45 Cys Asp Gln Glu Met Ala Gly Arg Ala Leu Thr GlnThr Gly Ser Arg 50 55 60 Ser Ile Glu Ala Ala Leu Glu Tyr Ile Ser Lys MetGly Tyr Leu Asp 65 70 75 80 Pro Arg Asn Glu Gln Ile Val Arg Val Ile LysGln Thr Ser Pro Gly 85 90 95 Lys Gly Leu Ala Ser Thr Pro Val Thr Arg ArgPro Ser Phe Glu Gly 100 105 110 Thr Gly Glu Ala Leu Pro Ser Tyr His GlnLeu Gly Gly Ala Asn Tyr 115 120 125 Glu Gly Pro Ala Ala Leu Glu Glu MetPro Arg Gln Tyr Leu Asp Phe 130 135 140 Leu Phe Pro Gly Ala Gly Ala GlyThr His Gly Ala Gln Ala His Gln 145 150 155 160 His Pro Pro Lys Gly TyrSer Thr Ala Val Glu Pro Ser Ala His Phe 165 170 175 Pro Gly Thr His TyrGly Arg Gly His Leu Leu Ser Glu Gln Ser Gly 180 185 190 Tyr Gly Val GlnArg Ser Ser Ser Phe Gln Asn Lys Thr Pro Pro Asp 195 200 205 Ala Tyr SerSer Met Ala Lys Ala Gln Gly Gly Pro Pro Ala Ser Leu 210 215 220 Thr PhePro Ala His Ala Gly Leu Tyr Thr Ala Ser His His Lys Pro 225 230 235 240Ala Ala Thr Pro Pro Gly Ala His Pro Leu His Val Leu Gly Thr Arg 245 250255 Gly Pro Thr Phe Thr Gly Glu Ser Ser Ala Gln Ala Val Leu Ala Pro 260265 270 Ser Arg Asn Ser Leu Asn Ala Asp Leu Tyr Glu Leu Gly Ser Thr Val275 280 285 Pro Trp Ser Ala Ala Pro Leu Ala Arg Arg Asp Ser Leu Gln LysGln 290 295 300 Gly Leu Glu Ala Ser Arg Pro His Val Ala Phe Arg Ala GlyPro Ser 305 310 315 320 Arg Thr Asn Ser Phe Asn Asn Pro Gln Pro Glu ProSer Leu Pro Ala 325 330 335 Pro Asn Thr Val Thr Ala Val Thr Ala Ala HisIle Leu His Pro Val 340 345 350 Lys Ser Val Arg Val Leu Arg Pro Glu ProGln Thr Ala Val Gly Pro 355 360 365 Ser His Pro Ala Trp Val Ala Ala ProThr Ala Pro Ala Thr Glu Ser 370 375 380 Leu Glu Thr Lys Glu Gly Ser AlaGly Pro His Pro Leu Asp Val Asp 385 390 395 400 Tyr Gly Gly Ser Glu ArgArg Cys Pro Pro Pro Pro Tyr Pro Lys His 405 410 415 Leu Leu Leu Pro SerLys Ser Glu Gln Tyr Ser Val Asp Leu Asp Ser 420 425 430 Leu Cys Thr SerVal Gln Gln Ser Leu Arg Gly Gly Thr Asp Leu Asp 435 440 445 Gly Ser AspLys Ser His Lys Gly Ala Lys Gly Asp Lys Ala Gly Arg 450 455 460 Asp LysLys Gln Ile Gln Thr Ser Pro Val Pro Val Arg Lys Asn Ser 465 470 475 480Arg Asp Glu Glu Lys Arg Glu Ser Arg Ile Lys Ser Tyr Ser Pro Tyr 485 490495 Ala Phe Lys Phe Phe Met Glu Gln His Val Glu Asn Val Ile Lys Thr 500505 510 Tyr Gln Gln Lys Val Ser Arg Arg Leu Gln Leu Glu Gln Glu Met Ala515 520 525 Lys Ala Gly Leu Cys Glu Ala Glu Gln Glu Gln Met Arg Lys IleLeu 530 535 540 Tyr Gln Lys Glu Ser Asn Tyr Asn Arg Leu Lys Arg Ala LysMet Asp 545 550 555 560 Lys Ser Met Phe Val Lys Ile Lys Thr Leu Gly IleGly Ala Phe Gly 565 570 575 Glu Val Cys Leu Ala Cys Lys Leu Asp Thr HisAla Leu Tyr Ala Met 580 585 590 Lys Thr Leu Arg Lys Lys Asp Val Leu AsnArg Asn Gln Val Ala His 595 600 605 Val Lys Ala Glu Arg Asp Ile Leu AlaGlu Ala Asp Asn Glu Trp Val 610 615 620 Val Lys Leu Tyr Tyr Ser Phe GlnAsp Lys Asp Ser Leu Tyr Phe Val 625 630 635 640 Met Asp Tyr Ile Pro GlyGly Asp Met Met Ser Leu Leu Ile Arg Met 645 650 655 Glu Val Phe Pro GluHis Leu Ala Arg Phe Tyr Ile Ala Glu Leu Thr 660 665 670 Leu Ala Ile GluSer Val His Lys Met Gly Phe Ile His Arg Asp Ile 675 680 685 Lys Pro AspAsn Ile Leu Ile Asp Leu Asp Gly His Ile Lys Leu Thr 690 695 700 Asp PheGly Leu Cys Thr Gly Phe Arg Trp Thr His Asn Ser Lys Tyr 705 710 715 720Tyr Gln Lys Gly Asn His Met Arg Gln Asp Ser Met Glu Pro Gly Asp 725 730735 Leu Trp Asp Asp Val Ser Asn Cys Arg Cys Gly Asp Arg Leu Lys Thr 740745 750 Leu Glu Gln Arg Ala Gln Lys Gln His Gln Arg Cys Leu Ala His Ser755 760 765 Leu Val Gly Thr Pro Asn Tyr Ile Ala Pro Glu Val Leu Leu ArgLys 770 775 780 Gly Tyr Thr Gln Leu Cys Asp Trp Trp Ser Val Gly Val IleLeu Phe 785 790 795 800 Glu Met Leu Val Gly Gln Pro Pro Phe Leu Ala ProThr Pro Thr Glu 805 810 815 Thr Gln Leu Lys Val Ile Asn Trp Glu Ser ThrLeu His Ile Pro Thr 820 825 830 Gln Val Arg Leu Ser Ala Glu Ala Arg AspLeu Ile Thr Lys Leu Cys 835 840 845 Cys Ala Ala Asp Cys Arg Leu Gly ArgAsp Gly Ala Asp Asp Leu Lys 850 855 860 Ala His Pro Phe Phe Asn Thr IleAsp Phe Ser Arg Asp Ile Arg Lys 865 870 875 880 Gln Ala Ala Pro Tyr ValPro Thr Ile Ser His Pro Met Asp Thr Ser 885 890 895 Asn Phe Asp Pro ValAsp Glu Glu Ser Pro Trp His Glu Ala Ser Gly 900 905 910 Glu Ser Ala LysAla Trp Asp Thr Leu Ala Ser Pro Ser Ser Lys His 915 920 925 Pro Glu HisAla Phe Tyr Glu Phe Thr Phe Arg Arg Phe Phe Asp Asp 930 935 940 Asn GlyTyr Pro Phe Arg Cys Pro Lys Pro Ser Glu Pro Ala Glu Ser 945 950 955 960Ala Asp Pro Gly Asp Ala Asp Leu Glu Gly Ala Ala Glu Gly Cys Gln 965 970975 Pro Val Tyr Val 980 6 amino acids amino acid unknown peptide 9 AspLeu Lys Pro Glu Asn 1 5 9 amino acids amino acid unknown peptide Peptide/label= A /note= “X at the second position can be either Threonine orSerine.” Peptide /label= B /note= “X at the fifth position can either beTyrosine or Phenylalanine.” 10 Gly Xaa Xaa Xaa Xaa Xaa Ala Pro Glu 1 5620 amino acids amino acid unknown protein 11 Met Asp Asn Thr Asn ArgPro His Leu Asn Leu Gly Thr Asn Asp Thr 1 5 10 15 Arg Met Ala Pro AsnAsp Arg Thr Tyr Pro Thr Thr Pro Ser Thr Phe 20 25 30 Pro Gln Pro Val PhePro Gly Gln Gln Ala Gly Gly Ser Gln Gln Tyr 35 40 45 Asn Gln Ala Tyr AlaGln Ser Gly Asn Tyr Tyr Gln Gln Asn His Asn 50 55 60 Asp Pro Asn Thr GlyLeu Ala His Gln Phe Ala His Gln Asn Ile Gly 65 70 75 80 Ser Ala Gly ArgAla Ser Pro Tyr Gly Ser Arg Gly Pro Ser Pro Ala 85 90 95 Gln Arg Pro ArgThr Ser Gly Asn Ser Gly Gln Gln Gln Thr Tyr Gly 100 105 110 Asn Tyr LeuSer Ala Pro Met Pro Ser Asn Thr Gln Thr Glu Phe Ala 115 120 125 Pro LeuPro Ser Gly Thr Pro Thr Asn Met Ala Pro Met Pro Thr Thr 130 135 140 ThrArg Arg Ser Ala His Ser Trp Pro Leu Thr Ser Leu Arg Thr Ala 145 150 155160 Ser Ser Ala Pro Gly Ser Ala Thr Arg Gly Glu Cys Cys Ser Asp Ala 165170 175 Leu Leu Pro Leu His Pro Ala Val Ile Gly Ala Asp Thr Leu Phe Arg180 185 190 Gln Ser Glu Met Glu Gln Lys Leu Gly Glu Thr Asn Asp Ala ArgArg 195 200 205 Arg Glu Ser Ile Trp Ser Thr Ala Gly Arg Lys Glu Gly GlnTyr Leu 210 215 220 Arg Phe Leu Arg Thr Lys Asp Lys Pro Glu Asn Tyr GlnThr Ile Lys 225 230 235 240 Ile Ile Gly Lys Gly Ala Phe Gly Glu Val LysLeu Val Gln Lys Lys 245 250 255 Ala Asp Gly Lys Val Tyr Ala Met Lys SerLeu Ile Lys Thr Glu Met 260 265 270 Phe Lys Lys Asp Gln Leu Ala His ValArg Ala Glu Arg Asp Ile Leu 275 280 285 Ala Glu Ser Asp Ser Pro Trp ValVal Lys Leu Tyr Thr Thr Phe Gln 290 295 300 Asp Ala Asn Phe Leu Tyr MetLeu Met Glu Phe Leu Pro Gly Gly Asp 305 310 315 320 Leu Met Thr Met LeuIle Lys Tyr Glu Ile Phe Ser Glu Asp Ile Thr 325 330 335 Arg Phe Tyr IleAla Glu Ile Val Leu Ala Ile Asp Ala Val His Lys 340 345 350 Leu Gly PheIle His Arg Asp Ile Lys Pro Asp Asn Ile Leu Leu Asp 355 360 365 Arg GlyGly His Val Lys Leu Thr Asp Phe Gly Leu Ser Thr Gly Phe 370 375 380 HisLys Leu His Asp Asn Asn Tyr Tyr Thr Gln Leu Leu Gln Gly Lys 385 390 395400 Ser Asn Lys Pro Arg Asp Asn Arg Asn Ser Val Ala Ile Asp Gln Ile 405410 415 Asn Leu Thr Val Ser Asn Arg Ala Gln Ile Asn Asp Trp Arg Arg Ser420 425 430 Arg Arg Leu Met Ala Tyr Ser Thr Val Gly Thr Pro Asp Tyr IleAla 435 440 445 Pro Glu Ile Phe Thr Gly His Gly Tyr Ser Phe Asp Cys AspTrp Trp 450 455 460 Ser Leu Gly Thr Ile Met Phe Glu Cys Leu Val Gly TrpPro Pro Phe 465 470 475 480 Cys Ala Glu Asp Ser His Asp Thr Tyr Arg LysIle Val Asn Trp Arg 485 490 495 His Ser Leu Tyr Phe Pro Asp Asp Ile ThrLeu Gly Val Asp Ala Glu 500 505 510 Asn Leu Ile Arg Ser Leu Ile Cys AsnThr Glu Asn Arg Leu Gly Arg 515 520 525 Gly Gly Ala His Glu Ile Lys SerHis Ala Phe Phe Arg Gly Val Glu 530 535 540 Phe Asp Ser Leu Arg Arg IleArg Ala Pro Phe Glu Pro Arg Leu Thr 545 550 555 560 Ser Ala Ile Asp ThrThr Tyr Phe Pro Thr Asp Glu Ile Asp Gln Thr 565 570 575 Asp Asn Ala ThrLeu Leu Lys Ala Gln Gln Ala Ala Arg Gly Ala Ala 580 585 590 Ala Pro AlaGln Gln Glu Glu Ser Pro Glu Leu Ser Leu Pro Phe Ile 595 600 605 Gly TyrThr Phe Lys Arg Phe Asp Asn Asn Phe Arg 610 615 620 526 amino acidsamino acid unknown protein 12 Met Asp Ser Ala Arg Gly Trp Phe Gln LysLeu Ser Ser Thr Lys Lys 1 5 10 15 Asp Pro Met Ala Ser Gly Arg Glu AspGly Lys Pro Val Ser Ala Glu 20 25 30 Glu Ala Ser Asn Ile Thr Lys Gln ArgVal Ala Ala Ala Lys Gln Tyr 35 40 45 Ile Glu Lys His Tyr Arg Glu Gln MetLys Asn Leu Gln Glu Arg Arg 50 55 60 Glu Arg Arg Ile Leu Leu Glu Lys LysLeu Ala Asp Ala Asp Val Ser 65 70 75 80 Glu Glu Asp Gln Asn Asn Leu LeuLys Phe Leu Glu Lys Lys Glu Thr 85 90 95 Glu Tyr Met Arg Leu Gln Arg HisLys Met Gly Ala Asp Asp Phe Glu 100 105 110 Leu Leu Thr Met Ile Gly LysGly Ala Phe Gly Glu Pro Ile Cys Met 115 120 125 Ile Gly Phe Ser Val IleThr Gly Gln Asn Cys Arg Glu Lys Thr Thr 130 135 140 Gly Gln Val Tyr AlaMet Lys Lys Leu Lys Lys Ser Glu Met Leu Arg 145 150 155 160 Arg Gly GlnVal Glu His Val Lys Ala Glu Arg Asn Leu Leu Ala Glu 165 170 175 Val AspSer Asp Cys Ile Val Lys Leu Tyr Tyr Ser Phe Gln Asp Asp 180 185 190 AspTyr Leu Tyr Leu Val Met Glu Tyr Leu Pro Gly Gly Asp Met Met 195 200 205Thr Leu Leu Met Arg Lys Asp Ile Leu Thr Glu Asp Glu Ala Arg Phe 210 215220 Tyr Val Ala Glu Thr Val Leu Ala Ile Glu Ser Ile His Lys His Asn 225230 235 240 Tyr Ile His Arg Asp Ile Lys Pro Asp Asn Leu Leu Leu Asp ArgTyr 245 250 255 Gly His Leu Lys Leu Ser Asp Phe Gly Leu Cys Lys Pro LeuAsp Cys 260 265 270 Ser Thr Leu Glu Glu Lys Asp Phe Ser Val Gly Asp AsnAla Asn Gly 275 280 285 Gly Ser Arg Ser Asp Ser Pro Pro Ala Pro Lys ArgThr Gln Gln Glu 290 295 300 Gln Leu Glu His Trp Gln Lys Asn Arg Arg MetLeu Ala Tyr Ser Thr 305 310 315 320 Val Gly Thr Pro Asp Tyr Ile Ala ProGlu Val Leu Leu Lys Lys Gly 325 330 335 Tyr Gly Met Glu Cys Asp Trp TrpSer Leu Gly Ala Ile Met Tyr Glu 340 345 350 Met Leu Val Gly Tyr Pro ProPhe Tyr Ser Asp Asp Pro Met Ser Thr 355 360 365 Cys Arg Lys Ile Val AsnTrp Lys Asn His Leu Lys Phe Pro Glu Glu 370 375 380 Ala Lys Leu Ser ProGlu Ala Lys Asp Ile Ile Ser Arg Leu Leu Cys 385 390 395 400 Asn Val ThrGlu Arg Leu Gly Ser Asn Gly Ala Asp Glu Ile Lys Val 405 410 415 His SerTrp Phe Lys Gly Ile Asp Trp Asp Arg Ile Tyr Gln Met Glu 420 425 430 AlaAla Phe Ile Pro Glu Val Asn Asp Glu Leu Asp Thr Gln Asn Phe 435 440 445Glu Lys Phe Glu Glu Ser Glu Ser His Ser Gln Ser Gly Ser Arg Ser 450 455460 Gly Pro Trp Arg Lys Met Leu Ser Ser Lys Asp Ile Asn Phe Val Gly 465470 475 480 Tyr Thr Tyr Lys Asn Phe Lys Val Val Asn Asp Tyr Gln Val ProGly 485 490 495 Met Val Glu Leu Lys Lys Thr Asn Thr Lys Pro Lys Lys ProThr Ile 500 505 510 Lys Ser Leu Phe Gly Asp Glu Ser Glu Ala Ser Glu AspAsn 515 520 525 479 amino acids amino acid unknown protein 13 Arg LysLeu His Asp Ala Asp Val Ser Glu Glu Asp Gln Asn Asn Leu 1 5 10 15 LeuLys Phe Leu Glu Lys Lys Glu Thr Glu Tyr Met Arg Leu Gln Arg 20 25 30 HisLys Met Gly Ala Asp Asp Phe Glu Leu Leu Thr Met Ile Gly Lys 35 40 45 GlyAla Phe Gly Glu Val Arg Val Cys Arg Glu Lys Thr Thr Gly His 50 55 60 ValTyr Ala Met Lys Lys Leu Lys Lys Ser Glu Met Leu Arg Arg Gly 65 70 75 80Gln Val Glu His Val Lys Ala Glu Arg Asn Leu Leu Ala Glu Val Asp 85 90 95Ser Asn Cys Ile Val Lys Leu Tyr Cys Ser Phe Gln Asp Glu Glu Tyr 100 105110 Leu Tyr Leu Ile Met Glu Tyr Leu Pro Gly Gly Asp Met Met Thr Leu 115120 125 Leu Met Arg Lys Asp Thr Leu Thr Glu Asp Glu Ala Arg Phe Tyr Val130 135 140 Ala Glu Thr Ile Leu Ala Ile Glu Ser Ile His Lys His Asn TyrIle 145 150 155 160 His Arg Asp Ile Lys Pro Asp Asn Leu Leu Leu Asp LysPhe Gly His 165 170 175 Leu Arg Leu Ser Asp Phe Gly Leu Cys Lys Pro LeuAsp Cys Ser Thr 180 185 190 Leu Glu Glu Lys Asp Phe Glu Val Asn Asn GlyAsn Gly Gly Ser Pro 195 200 205 Ser Asn Glu Gly Ser Thr Lys Pro Arg ArgThr Gln Gln Glu Gln Leu 210 215 220 Gln His Trp Gln Lys Asn Arg Arg MetLeu Ala Tyr Ser Thr Val Gly 225 230 235 240 Thr Pro Asp Tyr Ile Ala ProGlu Val Leu Leu Lys Lys Gly Tyr Gly 245 250 255 Met Glu Cys Asp Trp TrpSer Leu Gly Ala Ile Met Tyr Glu Met Leu 260 265 270 Val Gly Tyr Pro ProPhe Tyr Ser Asp Asp Pro Met Ser Thr Cys Arg 275 280 285 Lys Ile Val AsnTrp Arg Thr His Leu Lys Phe Pro Glu Glu Ala Lys 290 295 300 Leu Ser ProGlu Ala Lys Asp Leu Ile Ser Lys Leu Leu Cys Asn Val 305 310 315 320 ThrGln Arg Leu Gly Ser Asn Gly Ala His Glu Ile Lys Leu His Pro 325 330 335Trp Phe Asn Gly Ile Asp Trp Glu Arg Ile Tyr Gln Met Glu Ala Ala 340 345350 Phe Ile Pro Glu Val Asn Asp Glu Leu Asp Thr Gln Asn Phe Glu Lys 355360 365 Phe Glu Glu Ala Asp Asn Ser Ser Gln Ser Thr Ser Lys Ala Gly Pro370 375 380 Trp Arg Lys Met Leu Ser Ser Lys Asp Leu Asn Phe Val Gly TyrThr 385 390 395 400 Tyr Lys Asn Phe Glu Ile Val Asn Asp Tyr Gln Val ProGly Ile Ala 405 410 415 Glu Leu Lys Lys Lys Asp Thr Lys Pro Lys Arg ProSer Ile Lys Ser 420 425 430 Leu Phe Glu Asp Glu Ser Ser Asp Ser Ser GluAla Ala Thr Ser Gly 435 440 445 Asp Gln Ser Val Gln Gly Ser Phe Leu AsnLeu Leu Pro Pro Gln Leu 450 455 460 Glu Val Ser Gln Thr Gln Thr Glu ValPro Pro Pro Lys Phe Thr 465 470 475 500 amino acids amino acid unknownprotein 14 Met Glu Lys Val Lys Ala Ala Lys Lys Phe Ile Glu Asn His TyrArg 1 5 10 15 Ser Gln Met Lys Asn Ile Gln Glu Arg Lys Glu Arg Arg TrpVal Leu 20 25 30 Glu Lys Gln Leu Ala Ser Ser Asp Val Pro Glu Glu Glu GlnMet Ser 35 40 45 Leu Ile Lys Asp Leu Glu Arg Lys Glu Thr Glu Phe Met ArgLeu Lys 50 55 60 Arg Asn Arg Ile Cys Val Asn Asp Phe Glu Leu Leu Thr IleIle Gly 65 70 75 80 Arg Gly Ala Tyr Gly Glu Val Gln Leu Cys Arg Glu LysLys Ser Glu 85 90 95 Asn Ile Tyr Ala Met Lys Lys Leu Lys Lys Ser Glu MetLeu Ser Arg 100 105 110 Gly Gln Val Glu His Val Arg Ala Glu Arg Asn LeuLeu Ala Glu Val 115 120 125 Asp Ser His Cys Ile Val Lys Leu Phe Tyr SerPhe Gln Asp Ala Glu 130 135 140 Tyr Leu Tyr Leu Ile Met Glu Tyr Leu ProGly Gly Asp Met Met Thr 145 150 155 160 Leu Leu Met Arg Glu Asp Ile LeuThr Glu Lys Val Ala Lys Phe Tyr 165 170 175 Ile Ala Gln Ser Val Leu AlaIle Glu Ser Ile His Lys His Asn Tyr 180 185 190 Ile His Arg Asp Ile LysPro Asp Asn Leu Leu Leu Asp Lys Asn Gly 195 200 205 His Met Lys Leu SerAsp Phe Gly Leu Cys Lys Pro Leu Asp Cys Ala 210 215 220 Thr Leu Ser ThrIle Lys Glu Asn Glu Ser Met Asp Asp Val Ser Lys 225 230 235 240 Asn SerMet Asp Ile Asp Ala Ser Leu Pro Asp Ala Gly Asn Gly His 245 250 255 SerTrp Arg Ser Ala Arg Glu Gln Leu Gln His Trp Gln Arg Asn Arg 260 265 270Arg Lys Leu Ala Phe Ser Thr Val Gly Thr Pro Asp Tyr Ile Ala Pro 275 280285 Glu Val Leu Leu Lys Lys Gly Tyr Gly Met Glu Cys Asp Trp Trp Ser 290295 300 Leu Gly Ala Ile Met Tyr Glu Met Leu Val Gly Tyr Pro Pro Phe Tyr305 310 315 320 Ser Asp Asp Pro Ile Thr Thr Cys Arg Lys Ile Val His TrpArg His 325 330 335 Tyr Leu Lys Phe Pro Asp Asp Ala Lys Leu Thr Phe GluAla Arg Asp 340 345 350 Leu Ile Cys Arg Leu Leu Cys Asp Val Glu His ArgLeu Gly Thr Gly 355 360 365 Gly Ala Glu Gln Ile Lys Val His Ala Trp PheLys Asp Val Glu Trp 370 375 380 Asp Arg Leu Tyr Glu Thr Asp Ala Ala TyrLys Pro Gln Val Asn Gly 385 390 395 400 Glu Leu Asp Thr Gln Asn Phe MetLys Phe Asp Glu Ala Asn Pro Pro 405 410 415 Thr Pro Ser Arg Ser Gly SerGly Pro Ser Arg Lys Met Leu Thr Ser 420 425 430 Lys Asp Leu Ser Phe ValGly Tyr Thr Tyr Lys Asn Phe Asp Ala Val 435 440 445 Lys Gly Leu Lys HisSer Phe Asp Arg Lys Gly Ser Thr Ser Pro Lys 450 455 460 Arg Pro Ser LeuAsp Ser Met Phe Asn Glu Asn Gly Met Asp Tyr Thr 465 470 475 480 Ala LysHis Ala Glu Glu Met Asp Val Gln Met Leu Thr Ala Asp Asp 485 490 495 CysMet Ser Pro 500 564 amino acids amino acid unknown protein 15 Met PheSer Arg Ser Asp Arg Glu Val Asp Asp Leu Ala Gly Asn Met 1 5 10 15 SerHis Leu Gly Phe Tyr Asp Leu Asn Ile Pro Lys Pro Thr Ser Pro 20 25 30 GlnAla Gln Tyr Arg Pro Ala Arg Lys Ser Glu Asn Gly Arg Leu Thr 35 40 45 ProGly Leu Pro Arg Ser Tyr Lys Pro Cys Asp Ser Asp Asp Gln Asp 50 55 60 ThrPhe Lys Asn Arg Ile Ser Leu Asn His Ser Pro Lys Lys Leu Pro 65 70 75 80Lys Asp Phe His Glu Arg Ala Ser Gln Ser Lys Thr Gln Arg Val Val 85 90 95Asn Val Cys Gln Leu Tyr Phe Leu Asp Tyr Tyr Cys Asp Met Phe Asp 100 105110 Tyr Val Ile Ser Arg Arg Gln Arg Thr Lys Gln Val Leu Arg Tyr Leu 115120 125 Glu Gln Gln Arg Ser Val Lys Asn Val Ser Asn Lys Val Leu Asn Glu130 135 140 Glu Trp Ala Leu Tyr Leu Gln Arg Glu His Glu Val Leu Arg LysArg 145 150 155 160 Arg Leu Lys Pro Lys His Lys Asp Phe Gln Ile Leu ThrGln Val Gly 165 170 175 Gln Gly Gly Tyr Gly Gln Val Tyr Leu Ala Lys LysLys Asp Ser Asp 180 185 190 Glu Ile Cys Ala Leu Lys Ile Leu Asn Lys LysLeu Leu Phe Lys Leu 195 200 205 Asn Glu Thr Asn His Val Leu Thr Glu ArgAsp Ile Leu Thr Thr Thr 210 215 220 Arg Ser Asp Trp Leu Val Lys Leu LeuTyr Ala Phe Gln Asp Pro Glu 225 230 235 240 Ser Leu Tyr Leu Ala Met GluPhe Val Pro Gly Gly Asp Phe Arg Thr 245 250 255 Leu Leu Ile Asn Thr ArgIle Leu Lys Ser Gly His Ala Arg Phe Tyr 260 265 270 Ile Ser Glu Met PheCys Ala Val Asn Ala Leu His Glu Leu Gly Tyr 275 280 285 Thr His Arg AspLeu Lys Pro Glu Asn Phe Leu Ile Asp Ala Thr Gly 290 295 300 His Ile LysLeu Thr Asp Phe Gly Leu Ala Ala Gly Thr Val Ser Asn 305 310 315 320 GluArg Ile Glu Ser Met Lys Ile Arg Leu Glu Glu Val Lys Asn Leu 325 330 335Gln Phe Pro Ala Phe Thr Glu Arg Ser Ile Glu Asp Arg Ser Lys Ile 340 345350 Tyr His Asn Met Arg Lys Thr Glu Ile Asn Tyr Ala Asn Ser Met Val 355360 365 Gly Ser Pro Asp Tyr Met Ala Leu Glu Val Leu Glu Gly Lys Lys Tyr370 375 380 Asp Phe Thr Val Asp Tyr Trp Ser Leu Gly Cys Met Leu Phe GluSer 385 390 395 400 Leu Val Gly Tyr Thr Pro Phe Ser Gly Ser Ser Thr AsnGlu Thr Tyr 405 410 415 Glu Asn Leu Arg Tyr Trp Lys Lys Thr Leu Arg ArgPro Arg Thr Glu 420 425 430 Asp Arg Arg Ala Ala Phe Ser Asp Arg Thr TrpAsp Leu Ile Thr Arg 435 440 445 Leu Ile Ala Asp Pro Ile Asn Arg Val ArgSer Phe Glu Gln Val Arg 450 455 460 Lys Met Ser Tyr Phe Ala Glu Ile AsnPhe Glu Thr Leu Arg Thr Ser 465 470 475 480 Ser Pro Pro Phe Ile Pro GlnLeu Asp Asp Glu Thr Asp Ala Gly Tyr 485 490 495 Phe Asp Asp Phe Thr AsnGlu Glu Asp Met Ala Lys Tyr Ala Asp Val 500 505 510 Phe Lys Arg Gln AsnLys Leu Ser Ala Met Val Asp Asp Ser Ala Val 515 520 525 Asp Ser Lys LeuVal Gly Phe Thr Phe Arg His Arg Asp Gly Lys Gln 530 535 540 Gly Ser SerGly Ile Leu Tyr Asn Gly Ser Glu His Ser Asp Pro Phe 545 550 555 560 SerThr Phe Tyr 561 amino acids amino acid unknown protein 16 Met Ala GlyAsn Met Ser Asn Leu Ser Phe Asp Gly His Gly Thr Pro 1 5 10 15 Gly GlyThr Gly Leu Phe Pro Asn Gln Asn Ile Thr Lys Arg Arg Thr 20 25 30 Arg ProAla Gly Ile Asn Asp Ser Pro Ser Pro Val Lys Pro Ser Phe 35 40 45 Phe ProTyr Glu Asp Thr Ser Asn Met Asp Ile Asp Glu Val Ser Gln 50 55 60 Pro AspMet Asp Val Ser Asn Ser Pro Lys Lys Leu Pro Pro Lys Phe 65 70 75 80 TyrGlu Arg Ala Thr Ser Asn Lys Thr Gln Arg Val Val Ser Val Cys 85 90 95 LysMet Tyr Phe Leu Glu Tyr Tyr Cys Asp Met Phe Asp Tyr Val Ile 100 105 110Ser Arg Arg Gln Arg Thr Lys Gln Val Leu Glu Tyr Leu Gln Gln Gln 115 120125 Ser Gln Leu Pro Asn Ser Asp Gln Ile Lys Leu Asn Glu Glu Trp Ser 130135 140 Ser Tyr Leu Gln Arg Glu His Gln Val Leu Arg Lys Arg Arg Leu Lys145 150 155 160 Pro Lys Asn Arg Asp Phe Glu Met Ile Thr Gln Val Gly GlnGly Gly 165 170 175 Tyr Gly Gln Val Tyr Leu Ala Arg Lys Lys Asp Thr LysGlu Val Cys 180 185 190 Ala Leu Lys Ile Leu Asn Lys Lys Leu Leu Phe LysLeu Asn Glu Thr 195 200 205 Lys His Val Leu Thr Glu Arg Asp Ile Leu ThrThr Thr Arg Ser Glu 210 215 220 Trp Leu Val Lys Leu Leu Tyr Ala Phe GlnGlu Leu Gln Ser Leu Tyr 225 230 235 240 Leu Ala Met Glu Phe Val Pro GlyGly Asp Phe Arg Thr Leu Leu Ile 245 250 255 Asn Thr Arg Cys Leu Lys SerGly His Ala Arg Phe Tyr Ile Ser Glu 260 265 270 Met Phe Cys Ala Val AsnAla Leu His Asp Leu Gly Tyr Thr His Arg 275 280 285 Asp Leu Lys Pro GluAsn Phe Leu Ile Asp Ala Lys Gly His Ile Lys 290 295 300 Leu Thr Asp PheGly Leu Ala Ala Gly Thr Ile Ser Asn Glu Arg Ile 305 310 315 320 Glu SerMet Lys Ile Arg Leu Glu Lys Ile Lys Asp Leu Glu Phe Pro 325 330 335 AlaPhe Thr Glu Lys Ser Ile Glu Asp Arg Arg Lys Met Tyr Asn Gln 340 345 350Leu Arg Glu Lys Glu Ile Asn Tyr Ala Asn Ser Met Val Gly Ser Pro 355 360365 Asp Tyr Met Ala Leu Glu Val Leu Glu Gly Lys Lys Tyr Asp Phe Thr 370375 380 Val Asp Tyr Trp Ser Leu Gly Cys Met Leu Phe Glu Ser Leu Val Gly385 390 395 400 Tyr Thr Pro Phe Ser Gly Ser Ser Thr Asn Glu Thr Tyr AspAsn Leu 405 410 415 Arg Arg Trp Lys Gln Thr Leu Arg Arg Pro Arg Gln SerAsp Gly Arg 420 425 430 Ala Ala Phe Ser Asp Arg Thr Trp Asp Leu Ile ThrArg Leu Ile Ala 435 440 445 Asp Pro Ile Asn Arg Leu Arg Ser Phe Glu HisVal Lys Arg Met Ser 450 455 460 Tyr Phe Ala Asp Ile Asn Phe Ser Thr LeuArg Ser Met Ile Pro Pro 465 470 475 480 Phe Thr Pro Gln Leu Asp Ser GluThr Asp Ala Gly Tyr Phe Asp Asp 485 490 495 Phe Thr Ser Glu Ala Asp MetAla Lys Tyr Ala Asp Val Phe Lys Arg 500 505 510 Gln Asp Lys Leu Thr AlaMet Val Asp Asp Ser Ala Val Ser Ser Lys 515 520 525 Leu Val Gly Phe ThrPhe Arg His Arg Asn Gly Lys Gln Gly Ser Ser 530 535 540 Gly Ile Leu PheAsn Gly Leu Glu His Ser Asp Pro Phe Ser Thr Phe 545 550 555 560 Tyr

What is claimed is:
 1. An isolated nucleic acid molecule that hybridizesunder conditions of low stringency to a second nucleic acid moleculeconsisting of a sequence selected from the group consisting of SEQ IDNO:1, the complement of SEQ ID NO: 1, SEQ ID NO:3, the complement of SEQID NO:3, SEQ ID NO:5, the complement of SEQ ID NO:5, SEQ ID NO: 7, andthe complement of SEQ ID NO:7, said conditions of low stringencyconsisting of pretreatment for 6 hours at 40° C. in a solutioncontaining 35% formamide, 5×SSC, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1%PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA;hybridization for 18-20 hours at 40° C. in a solution containing 35%formamide, 5×SSC, 550OmM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02%Ficoll, 0.2% BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (wt/vol)dextran sulfate; and washing twice for 1.5 hours at 55° C. in a solutioncontaining 2×SSC, 25 mM Tris-HC (pH7.4), 5 mM EDTA, and 0.1% SDS,wherein said isolated nucleic acid encodes, or is complementary to asequence that encodes, a lats polypeptide or a fragment thereofcomprising one or more lats domains selected from the group consistingof a lats C-terminal domain 3 (LCD3), lats C-terminal domain 2 (LCD2),lats C-terminal domain 1 (LCD1), kinase domain, lats flanking domain(LFD), lats split domain 1 (LSD1), lats split domain 2 (LSD2),SH3-binding domain, and opa repeat domain, and wherein the isolated latspolypeptide or fragment thereof has a functional activity of afull-length lats protein.
 2. The isolated nucleic acid molecule of claim1, wherein said polypeptide comprises the amino acid sequence of SEQ IDNO:2, or a domain-containing fragment thereof.
 3. The isolated nucleicacid molecule of claim 1, wherein said polypeptide comprises the aminoacid sequence of SEQ ID NO:4, or a domain-containing fragment thereof.4. The isolated nucleic acid molecule of claim 1, wherein saidpolypeptide comprises the amino acid sequence selected from the groupconsisting of SEQ ID NO:6 and SEQ ID NO:8, or a domain containingfragment thereof.
 5. An isolated vector comprising the nucleic acidmolecule of claim
 1. 6. A recombinant cell in vitro comprising thevector of claim
 5. 7. A method of producing a lats polypeptidecomprising growing the recombinant cell of claim 5 in culture underconditions in which said polypeptide is expressed by the cell, andrecovering said polypeptide.
 8. A cell transformed in vitro with thevector of claim
 5. 9. A transgenic Drosophila whose germ cells comprisea nucleic acid molecule encoding a lats protein kinase operably linkedto a glass promoter, wherein said nucleic acid molecule encoding thelats protein kinase is expressed in the posterior region of thedeveloping third instar larval eve imaginal disc of said Drosophila, andwherein said Drosophila has a phenotype of small-rough eyes.